Influenza virus vaccines and uses thereof

ABSTRACT

Provided herein are influenza hemagglutinin stem domain polypeptides, methods for providing hemagglutinin stem domain polypeptides, compositions comprising the same, vaccines comprising the same and methods of their use, in particular in the detection, prevention and/or treatment of influenza

INTRODUCTION

The invention relates to the field of medicine. Provided herein areinfluenza hemagglutinin stem domain polypeptides, methods for providinghemagglutinin stem domain polypeptides, compositions comprising thesame, vaccines comprising the same and methods of their use, inparticular in the detection, prevention and/or treatment of influenza.

BACKGROUND

Influenza viruses are major human pathogens, causing a respiratorydisease (commonly referred to as “influenza” or “the flu”) that rangesin severity from sub-clinical infection to primary viral pneumonia whichcan result in death. The clinical effects of infection vary with thevirulence of the influenza strain and the exposure, history, age, andimmune status of the host. Every year it is estimated that approximately1 billion people worldwide undergo infection with influenza virus,leading to severe illness in 3-5 million cases and an estimated 300,000to 500,000 of influenza related deaths. The bulk of these infections canbe attributed to influenza A viruses carrying H1 or H3 hemagglutininsubtypes, with a smaller contribution from Influenza B viruses, andtherefore representatives of all three are included in the seasonalvaccine. The current immunization practice relies on earlyidentification of circulating influenza viruses to allow for timelyproduction of an effective seasonal influenza vaccine. Apart from theinherent difficulties in predicting the strains that will be dominantduring the next season, antiviral resistance and immune escape also playa role in failure of current vaccines to prevent morbidity andmortality. In addition to this the possibility of a pandemic caused by ahighly virulent viral strain originating from animal reservoirs andreassorted to increase human to human spread, poses a significant andrealistic threat to global health.

Influenza A viruses are widely distributed in nature and can infect avariety of birds and mammals. Influenza viruses are enveloped RNAviruses that belong to the family of Orthomyxoviridae. Their genomesconsist of eight single-stranded RNA segments that code for 11 differentproteins, one nucleoprotein (NP), three polymerase proteins (PA, PB1,and PB2), two matrix proteins (M1 and M2), three non-structural proteins(NS1, NS2, and PB1-F2), and two external glycoproteins: hemagglutinin(HA) and neuraminidase (NA). The viruses are classified on the basis ofdifferences in antigenic structure of the HA and NA proteins, with theirdifferent combinations representing unique virus subtypes that arefurther classified into specific influenza virus strains. Although allknown subtypes can be found in birds, currently circulating humaninfluenza A subtypes are H1N1 and H3N2. Phylogenetic analysis hasdemonstrated a subdivision of hemagglutinins into two main groups: interalia the H1, H2, H5 and H9 subtypes in phylogenetic group 1 and interalia the H3, H4 and H7 subtypes in phylogenetic group 2.

The influenza type B virus strains are strictly human. The antigenicvariation in HA within the influenza type B virus strains is smallerthan those observed within the type A strains. Two genetically andantigenically distinct lineages of influenza B virus are circulating inhumans, as represented by the B/Yamagata/16/88 (also referred to asB/Yamagata) and BNictoria/2/87 (BNictoria) lineages (Ferguson et al.,2003). Although the spectrum of disease caused by influenza B viruses isgenerally milder than that caused by influenza A viruses, severe illnessrequiring hospitalization is still frequently observed with influenza Binfection.

It is known that antibodies that neutralize the influenza virus areprimarily directed against hemagglutinin (HA). Hemagglutinin or HA is atrimeric glycoprotein that is anchored to the viral coat and has a dualfunction: it is responsible for binding to the cell surface receptorsialic acid and, after uptake, it mediates the fusion of the viral andendosomal membrane leading to release of the viral RNA in the cytosol ofthe cell. HA comprises a large head domain and a smaller stem domain.Attachment to the viral membrane is mediated by a C-terminal anchoringsequence connected to the stem domain. The protein ispost-translationally cleaved in a designated loop to yield twopolypeptides, HA1 and HA2 (the full sequence is referred to as HA0). Themembrane distal head region is mainly derived from HA1 and the membraneproximal stem region primarily from HA2 (FIG. 1).

The reason that the seasonal influenza vaccine must be updated everyyear is the large variability of the virus. In the hemagglutininmolecule this variation is particularly manifested in the head domainwhere antigenic drift and shift have resulted in a large number ofdifferent variants. Since this is also the area that is immunodominant,most neutralizing antibodies are directed against this domain and act byinterfering with receptor binding. The combination of immunodominanceand large variation of the head domain also explains why infection witha particular strain does not lead to immunity to other strains: theantibodies elicited by the first infection only recognize a limitednumber of strains closely related to the virus of the primary infection.

Recently, influenza hemagglutinin stem domain polypeptides, lacking allor substantially all of the influenza hemagglutinin globular headdomain, have been described and used to generate an immune response toone or more conserved epitopes of the stem domain polypeptide. It isbelieved that epitopes of the stem domain polypeptide are lessimmunogenic than the highly immunogenic regions of a globular headdomain, thus the absence of a globular head domain in the stem domainpolypeptide might allow an immune response against one or more epitopesof the stem domain polypeptide to develop (Steel et al., 2010). Steel etal. thus have created a new molecule by deleting amino acid residue 53to 276 of HA1 of the A/Puerto Rico/8/1934 (H1N1) and A/Hong Kong/1968(H3N2) strains from the HA primary sequence, and replacing this by ashort flexible linking sequence GGGG. Vaccination of mice with the H3HK68 construct did not elicit antisera that were cross-reactive withgroup 1 HAs. In addition, as shown in PCT/EP2012/073706, the stem domainpolypeptides were highly unstable and did not adopt the correctconformation as proven by the lack of binding of antibodies that wereshown to bind to conserved epitopes in the stem region.

In addition, Bommakanti et al. (2010) described an HA2 based polypeptidecomprising amino acid residues 1-172 of HA2, a 7-amino acid linker(GSAGSAG), amino acid residues 7-46 of HA1, a 6-amino acid linkerGSAGSA, followed by residues 290-321 of HA1, with the mutations V297T,1300E, Y302T and C305T in HA1. The design was based on the sequence ofH3 HA (A/Hong Kong/1968). The polypeptide did only providecross-protection against another influenza virus strain within the H3subtype (A/Phil/2/82 but not against an H1 subtype (A/PR/8/34). In amore recent paper by Bommakanti et al (2012) a stem domain sequencebased on HA from H1N1 A/Puerto Rico/8/1934 (HIHAOHA6) is described. Inthis polypeptide the equivalent of residues 55 to 302 have been deletedand mutations 1311T, V314T, 1316N, C319S, F406D, F409T, and L416D havebeen made. Both the H3 and HA based polypeptides were expressed in E.coli and therefore lack the glycans that are part of the naturallyoccurring HA proteins. When expressed in E. coli the polypeptide isrecovered mainly as high molecular weight aggregates and a minormonomeric fraction. The polypeptide binds CR6261 with two apparentdissociation constants of 9 and 0.2 μM. The authors show that mice cansurvive a challenge with 1 LD90 of the homologous H1N1 A/PuertoRico/8/1934 virus after immunization (twice, 4 week interval) with 20 μgof protein adjuvanted with 100 μg of CpG7909. The authors also describecircularly permutated polypeptides comparable to those described abovefor A/Hong Kong/1/1968 derived polypeptides. These polypeptides arederived from HA's from H1N1 A/Puerto Rico/8/1934, H1N1 A/NorthCarolina/20/99 or H1N1 A/California/07/2009 and can provide partialprotection in a mild challenge (1LD90) model in mice of H1N1 A/PuertoRico/8/1934 (i.e. within the same subtype). Sera from guinea pigsimmunized with these polypeptides did not exhibit detectable levels ofneutralization when tested in a neutralization assay. More recently Luet al (2013) also described soluble stem domain polypeptides derivedfrom the HA of H1N1 A/California/05/2009. In the final design theequivalent of residues 54-303 (numbering according to SEQ ID NO: 1) havebeen deleted (the leader sequence, residues 1-17 is also not present)and two mutations have been introduced in the B-loop of the protein,i.e. F407D, and L413D. Furthermore the polypeptide contained aC-terminal trimerization domain (foldon). In addition, two intermonomerdisulfide bridges were introduced, one in the area of the trimericfoldon domain, and one at position 430 and 431. The polypeptide isproduced in an E. coli based cell free system, (and thus lacks theglycans that arepart of the naturally occurring HA proteins) and isrecovered in a denatured form, which needs to be refolded prior to use.No immunological or protection from influenza challenge data were shown.

In a recent paper Mallajosyula et al (2014) also report a stem domainpolypeptide. In this design, based on the HA from H1N1 A/PuertoRico/8/1934, not only a large part of the HA1 sequence is deleted(residue 42 to 289, numbering according to SEQ ID NO: 1), but also largepart of the N- and C-terminal sequences of HA2 (residues 344 to 383 and457 to 565, respectively). The polypeptide contains a foldontrimerization domain at the C-terminus and is also produced in E. coli,so lacks the naturally occurring glycans on viral HA. The polypeptidebinds the broadly neutralizing antibodies CR6261, F10 and FI6v3. Thepolypeptide was also tested in an influenza challenge model (1LD90 ofH1N1 A/Puerto Rico/8/1934) and could protect mice from death. Equivalentpolypeptides derived from HA of H1N1 A/New Caledonia/20/1999 and H1N1A/California/04/2009 could also partially protect. A polypeptide derivedfrom H5N1 A/Viet Nam/1203/2004 only gave limited protection in thischallenge model. Moreover, the challenge model used is mild with arelatively low dose administered (1-2 LD90).

There thus still exists a need for a safe and effective universalvaccine that stimulates the production of a robust, broadly neutralizingantibody response and that offers protection against a broad set ofcurrent and future influenza virus strains (both seasonal and pandemic),in particular providing protection against one or more influenza A virussubtypes within phylogenetic group 1 and/or group 2, for effectiveprevention and therapy of influenza.

SUMMARY

Provided herein are influenza hemagglutinin stem domain polypeptides,methods for providing stem domain polypeptides, compositions comprisingthe same, vaccines comprising the same and methods of their use.

In a first aspect, the present invention provides novel immunogenicpolypeptides comprising an influenza hemagglutinin stem domain andlacking the globular head, referred to as influenza hemagglutinin (HA)stem domain polypeptides. The polypeptides are capable of inducing animmune response when administered to a subject, in particular a humansubject. The polypeptides of the invention present conserved epitopes ofthe membrane proximal stem domain HA molecule to the immune system inthe absence of dominant epitopes that are present in the membrane distalhead domain. To this end, part of the primary sequence of the HA0protein making up the head domain is removed and the remaining aminoacid sequence is reconnected, either directly or, in some embodiments,by introducing a short flexible linking sequence (‘linker’) to restorethe continuity of the amino acid chain. The resulting sequence isfurther modified by introducing specific mutations that stabilize thenative 3-dimensional structure of the remaining part of the HA0molecule. The immunogenic polypeptides do not comprise the full-lengthHA1 domain of an influenza virus.

The present invention provides novel influenza hemagglutinin stem domainpolypeptide comprising: (a) an influenza hemagglutinin HA1 domain thatcomprises an HA1 N-terminal stem segment, covalently linked by a linkingsequence of 0-50 amino acid residues to an HA1 C-terminal stem segment,said HA1 C-terminal segment being linked to (b) an influenzahemagglutinin HA2 domain, wherein the HA1 and HA2 domain are derivedfrom an influenza A virus subtype comprising HA of the H1 subtype; and

(c) wherein the polypeptide comprises no protease cleavage site at thejunction between the HA1 domain and HA2 domain;(d) wherein said HA1 N-terminal segment comprises the amino acids 1-x ofHA1, preferably the amino acids p-x of HA1, and wherein the HA1C-terminal stem segment comprises the amino acids y-C-terminal aminoacid of HA1, wherein x=the amino acid on position 52 of SEQ ID NO: 1 (oran equivalent position in another hemagglutinin), p=the amino acid onposition 18 of SEQ ID NO: 1 (or an equivalent position in anotherhemagglutinin) and y=the amino acid on position 321 of SEQ ID NO: 1 (oran equivalent position in another hemagglutinin);(e) wherein the region comprising the amino acid residues 402-418comprises the amino acid sequence X₁NTQX₂TAX₃GKEX₄N(H/K)X₈E(K/R) (SEQ IDNO: 8), wherein:X₁ is an amino acid selected from the group consisting of M, E, K, V, Rand T,X₂ is an amino acid selected from the group consisting of F, I. N, T, H,L and Y, preferably I, L or Y,X₃ is an amino acid selected from the group consisting of V, A, G, I, R,F and S, preferably A, I or F,X₄ is an amino acid selected from the group consisting of F, I, N, S, T,Y, E, K, M, and V, preferably I, Y, M or V,X₅ is an amino acid selected from the group consisting of L, H, I, N, R,preferably I;(f) wherein the amino acid residue on position 337 (HA1 domain) isselected from the group consisting of: I, E, K, V, A, and T,the amino acid residue on position 340 (HA1 domain) is selected from thegroup consisting of: I, K, R, T, F, N, S and Y,the amino acid residue on position 352 (HA2 domain) is selected from thegroup consisting of: D, V, Y, A, I, N, S, and T, andthe amino acid residue on position 353 (HA2 domain) is selected from thegroup consisting of: K, R, T, E. G, and V; and(g) wherein the polypeptide further comprises a disulfide bridge betweenthe amino acid on position 324 and the amino acid on position 436; and(h) wherein furthermore the amino acid sequence RMKQIEDKIEEIESK (SEQ IDNO: 20) has been introduced at positions 419-433 or wherein sequenceRMKQIEDKIEEIESKQK (SEQ ID NO: 21) has been introduced at position417-433.

In certain embodiments, the polypeptides comprise the complete HA2domain, i.e. the HA2 domain including the transmembrane domain and theintracellular sequence. In certain embodiments, the HA2 domain has beentruncated. Thus, in certain embodiments, the polypeptides of theinvention do not contain the intracellular sequences of HA and thetransmembrane domain. In certain embodiments, the amino acid sequencefrom position (or the equivalent of) 514, 515, 516, 517, 518, 519, 520,521, 522, 523, 524, 525, 526, 527, 526, 528, 529, or 530 of the HA2domain to the C-terminus of the HA2 domain has been removed.

According to the invention, the C-terminal amino acid of the HA1C-terminal stem segment is linked to the N-terminal amino acid of theHA2 domain, thus forming a junction between the HA1 and HA2 domain. Thepolypeptides of the invention do not comprise a protease cleavage siteat the junction between the HA1 and HA2 domain. In certain embodiments,the C-terminal amino acid residue of the HA1 C-terminal stem segment(amino acid 343 in SEQ ID NO: 1) is any amino acid other than arginine(R) or lysine (K), preferably glutamine (Q).

The polypeptides of the invention are substantially smaller than HA0,preferably lacking all or substantially all of the globular head of HA.Preferably, the immunogenic polypeptides are no more than 360,preferably no more than 350, 340, 330, 320, 310, 305, 300, 295, 290,285, 280, 275, or 270 amino acids in length. In certain embodiments, theimmunogenic polypeptides are from about 250 to about 350, preferablyfrom about 260 to about 340, preferably from about 270 to about 330,preferably from about 270 to about 330 amino acids in length.

The polypeptides of the invention comprise the conserved stem domainepitopes of the group 1 cross-neutralizing antibody CR6261 (as disclosedin WO2008/028946) and/or of the antibody CR9114 (as described inWO2013/007770), an antibody capable of binding to and neutralizing bothgroup 1 and group 2 influenza A viruses, as well as influenza B viruses.It is thus another aspect of the invention to provide HA stem domainpolypeptides, wherein said polypeptides stably present the epitopes ofthe antibody CR6261 and/or CR9114, as indicated by binding of saidantibody or antibodies to said polypeptides. In an embodiment, thepolypeptides do not bind to CR8020 and CR8057 (described in WO2010/130636), which are monoclonal antibodies that binds to H3 influenzaviruses only.

The influenza hemagglutinin stem domain polypeptides provided herein aresuitable for use in immunogenic compositions (e.g. vaccines) capable ofgenerating immune responses against one/or a plurality of influenzavirus A and/or B strains, in particular against an influenza virus ofthe H1 subtype. In an embodiment, the influenza hemagglutinin stemdomain polypeptides are capable of generating immune responses againstinfluenza A virus strains of phylogenetic group 1 and/or group 2, inparticular against influenza virus strains of both phylogenetic group 1and group 2. In an embodiment, the polypeptides are capable ofgenerating an immune response against homologous influenza virusstrains. In an embodiment, the polypeptides are capable of generating animmune response against heterologous influenza virus strains of the sameand/or different subtypes. In a further embodiment, the polypeptides arecapable of generating an immune response to influenza virus strains ofboth phylogenetic group 1 and group 2 and influenza B virus strains.

The polypeptides according to the invention may be used e.g. in standalone therapy and/or prophylaxis and/or diagnosis of a disease orcondition caused by an influenza virus, in particular a phylogeneticgroup 1 or 2 influenza A virus and/or an influenza B virus, or incombination with other prophylactic and/or therapeutic treatments, suchas (existing or future) vaccines, antiviral agents and/or monoclonalantibodies.

In a further aspect, the present invention provides nucleic acidmolecules encoding the influenza HA stem domain polypeptides. In yetanother aspect, the invention provides vectors comprising the nucleicacids encoding the immunogenic polypeptides.

In a further aspect, the invention provides methods for inducing animmune response in a subject, the method comprising administering to thesubject a polypeptide and/or nucleic acid molecule and/or vectoraccording to the invention.

In another aspect, the invention provides compositions comprising apolypeptide and/or a nucleic acid molecule and/or a vector according tothe invention. The compositions preferably are immunogenic compositions.The compositions provided herein can be in any form that allows for thecompositions to be administered to a subject, e.g. mice, ferrets orhumans. In a specific embodiment, the compositions are suitable forhuman administration. The polypeptides, nucleic acid molecules andcompositions may be used in methods of preventing and/or treating aninfluenza virus disease and/or for diagnostic purposes. The compositionsmay further comprise a pharmaceutically acceptable carrier or excipient.In certain embodiments, the compositions described herein comprise, orare administered in combination with, an adjuvant.

In another aspect, the invention provides polypeptides, nucleic acidsand/or vectors for use as a vaccine. The invention in particular relatesto immunogenic polypeptides, nucleic acids, and/or vectors for use as avaccine in the prevention and/or treatment of a disease or conditioncaused by an influenza virus A subtype of phylogenetic group 1 and/or 2and/or influenza B virus, in particular a disease or condition caused byan influenza virus comprising HA of the H1 subtype.

The various embodiments and uses of the polypeptides according to theinvention will become clear from the following detailed description ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a model of the HA monomer in the pre-fusion state aspresent in the native trimer. HA 1 is shown in light grey, HA2 is shownin dark grey. Helix A (an important part of the epitope of CR6261) andhelix CD (part of the trimer interface) are indicated, as is the loopconnecting these secondary structure elements. The C-terminus of HA1 andthe N-terminus of HA2 are also indicated. The fusion peptide is locatedat the N-terminus of HA2.

FIG. 2. Sandwich Elisa results obtained for supernatants of culturesexpressing SEQ ID NO: 65 to 71 and SEQ ID NO: 76 to 78, disclosed inPCT/EP2014/060997. Capture and detection antibodies are indicated abovethe graph. Mini-HA refers to a soluble version of SEQ ID NO: 2 where theequivalent of residue 519-565 has been replaced by RSLVPRGSPGHHHHHH;FL-HA-FFH refers to a soluble version of SEQ ID NO: 1 containing aC-terminal Flag-thrombin-foldon-His sequence (SEQ ID NO: 4) fromposition 520; FL-HA-7×His refers to a soluble version of SEQ ID NO: 1containing the C-terminal sequence EGRHHHHHHH from position 530.

FIG. 3. Sandwich Elisa results obtained for supernatants of culturesexpressing polypeptides of the invention comprising GCN4 derivedsequence RMKQIEDKIEEIESK (SEQ ID NO: 20) at position 419-433 (t2variants). Capture and detection antibodies are indicated above thegraph. Mini-HA-t2 is derived from Mini-HA by introducing SEQ ID NO: 20at position 419-433; FL-HA-FFH, FL-HA-7×His as above.

FIG. 4. Sandwich Elisa results obtained for supernatants of culturesexpressing polypeptides of the invention comprising GCN4 derivedsequence RMKQIEDKIEEIESKQK (SEQ ID NO: 21) at position 417-433 (t3variants). Capture and detection antibodies are indicated above thegraph. Mini-HA-t3 is derived from Mini-HA by introducing SEQ ID NO: 21at position 417-433; FL-HA-FFH, FL-HA-7×His as above.

FIG. 5. Elution profiles of s55G7-t2, s127H1-t2 and s86B4-t2 from aSuperdex 200 size exclusion column, the final step in the purificationprocedure. The numbered lines under the chromatogram indicate fractionscollected during the elution process.

FIG. 6. SDS-PAGE and Western Blot analysis of fractions collected duringthe elution of the Superdex 200 size exclusion column. Numberscorrespond to the fractions indicated in FIG. 5. For detection onWestern Blot an antibody recognizing the C-terminal his-tag was used.

FIG. 7. Size exclusion chromatography (Tosoh G2000 analytical column) ofs127H1-t2 in the presence and absence of Fab fragments of broadlyneutralizing antibodies CR9114, CR6261, and CR8020. Molecular weights ofindividual proteins and/or complexes were determined by multi-anglelight scattering during elution from the column and are listed in Table8.

FIG. 8, Binding of polypeptide of the invention s127H1-t2 to monoclonalantibodies CR6261 and CR9114 using biolayer interferometry. Top panelsshow individual binding curves for immobilized monoclonal antibodiesexposed to varying concentrations of s127H1-t2, bottom panels show thesteady state analysis used to estimate K_(d).

FIG. 9. Survival (A), body weight loss (B) and clinical score (C) forthe negative (PBS, 3 immunizations at 3 weeks intervals) and positivecontrol (15 mg/kg CR6261, 1 day before challenge) groups. Mice werechallenged four week after the last immunization with a lethal dose(25×LD50) of H1N1 A/Puerto Rico/8/34 and monitored for 21 days. Errorbars indicate 95% confidence interval (B) or interquartile range (C)

FIG. 10. Survival (A), body weight loss (B) and clinical score (C) forthe experimental groups immunized (3 immunizations at 3 weeks intervals)with 10 μg s127H1-t2, either in the presence or absence of 10 μgMatrix-M. Mice were challenged four week after the last immunizationwith a lethal dose (25×LD50) of H1N1 A/Puerto Rico/8/34 and monitoredfor 21 days or reasons of comparison the negative control group (PBS) isalso shown. Error bars indicate 95% confidence interval (B) orinterquartile range (C) FIG. 11. Elisa results for serum of the negativecontrol and experimental groups using s127H1-t2 (A) or a soluble form ofFull length HA (B) as the antigen. Bars represent median.

FIG. 12. The antibodies induced after immunization with adjuvatedpolypeptide of the invention s127H1-t2 are capable of competing withCR9114 for binding to full length HA from H1N1 A/Brisbane/59/07 in acompetition ELISA (A). For reasons of comparison competition levels byunlabeled CR9114 (i.e. self-competition) and the non-binding monoclonalantibodies CR8020 and CR-JB, both serially diluted from 5 μg/ml startingconcentration, are indicated in a separate graph.

FIG. 13. Survival (A), relative body weight loss (B) and clinical score(C) for the negative (PBS, 3 immunizations at 3 weeks intervals) andpositive control (15 mg/kg CR6261, 1 day before challenge) groups. Micewere challenged four week after the last immunization with a lethal dose(25×LD50) of H1N1 A/Puerto Rico/8/34 and monitored for 21 days. Errorbars indicate 95% confidence interval (B) or interquartile range (C).

FIG. 14. Survival for groups immunized 1 time (A), 2 times (B) or 3times (C) with 30 μg s127H1-t2-c118long in the presence of 10 μgMatrix-M. Mice were challenged four week after the last immunizationwith a lethal dose (25×LD50) of H1N1 A/Puerto Rico/8/34 and monitoredfor 21 days. For reasons of comparison the negative control group (PBS)is also shown.

FIG. 15. Relative body weight change for groups immunized 1 time (A), 2times (B) or 3 times with 30 μg s127H1-t2-c118long in the presence of 10μg Matrix-M. Mice were challenged four week after the last immunizationwith a lethal dose (25×LD50) of H1N1 A/Puerto Rico/8/34 and monitoredfor 21 days. For reasons of comparison the negative control group (PBS)is also shown. Error bars indicate 95% confidence interval.

FIG. 16. Clinical scores for groups immunized 1 time (A), 2 times (B) or3 times with 30 μg s127H1-t2-c118long in the presence 10 μg Matrix-M.Mice were challenged four week after the last immunization with a lethaldose (25×LD50) of H1N1 A/Puerto Rico/8/34 and monitored for 21 days. Forreasons of comparison the negative control group (PBS) is also shown.Error bars indicate interquartile range.

FIG. 17. ELISA results for pre-challenge serum (4 weeks after the finalimmunization) of the negative control and experimental groups usings127H1-t2-c118long (A) or a soluble form of Full length HA (B) as theantigen. Bars represent median.

FIG. 18. The antibodies induced after immunization with Matrix-Madjuvated polypeptide of the invention s127H1-t2-c118long are capable ofcompeting with CR9114 for binding to full length HA from H1N1A/Brisbane/59/07 in a competition ELISA (A). For reasons of comparisoncompetition levels by unlabeled CR9114 (i.e. self-competition) and thenon-binding monoclonal antibodies CR8020, both serially diluted from 5μg/ml starting concentration, are indicated in a separate graph (B).Bars represent median.

FIG. 19. (A) Survival for the negative (PBS, 3 immunizations at 3 weeksintervals) and positive control (15 mg/kg CR6261, 1 day beforechallenge) groups. Mice were challenged four week after the lastimmunization with a lethal dose (12.5×LD50) of H5N1 A/Hong Kong/156/97.(B) Survival, (C) relative body weight change and (D) median clinicalscores for the group immunized 3 times with 30 μg s127H1-t2 in thepresence of 10 μg Matrix-M. Error bars indicate 95% confidence interval(C) or interquartile range (D). Mice were challenged four week after thelast immunization with a lethal dose (12.5×LD50) of H5N1 A/HongKong/156/97 and monitored for 21 days. For reasons of comparison thenegative control group (PBS) is also shown in B, C, D.

FIG. 20. Elisa results for sera from mice immunized 3 times withpolypeptide of the invention s127H1-t2 as described in example 5 usingfull length HA's from a number of group 1 (H1, H5 and H9) and group II(H3 and H7) influenza strains as the antigen. Induced antibodiesrecognize all tested FL HA's from group 1.

FIG. 21. (A) Survival for the negative (PBS, 3 immunizations at 3 weeksintervals) and positive control (15 mg/kg CR6261, 1 day beforechallenge) groups. Mice were challenged four week after the lastimmunization with a lethal dose (12.5×LD50) of H1N1 A/Brisbane/59/2007.(B) Survival, (C) relative body weight change and (D) median clinicalscores for the group immunized 3 times with 30 μg s127H1-t2 in thepresence of 10 μg Matrix-M. Error bars indicate 95% confidence interval(C) or interquartile range (D). Mice were challenged four week after thelast immunization with a lethal dose (12.5×LD50) of H1N1A/Brisbane/59/2007 and monitored for 21 days. For reasons of comparisonthe negative control group (PBS) is also shown in B, C, D.

FIG. 22. Pseudoparticle neutralizations assay using sera from miceimmunized with polypeptide of the invention s127H1-t2 or PBS.

FIG. 23. Antibody Dependent Cellular Cytotoxicity (ADCC) surrogateassay. Sera from mice immunized with polypeptide of the inventions127H1-t2 exhibit a 30-40 fold induction of FcγRIV signaling activity atthe highest serum concentrations using target cells transfected with FLHA from H5N1 A/Hong Kong/156/97 (A) or H1N1 A/Brisbane/59/07 (B) as thesource of antigen.

FIG. 24. Survival (A) and % body weight change (B) of mice after serumtransfer and challenge with H5N1 A/Hong Kong/156/97 as described inExample 9.

FIG. 25. Full length HA (H1N1 A/Brisbane/59/2007) ELISA titers of donormice (D) at day 70, and recipient mice (R) prior to serum transfer (day−4) or challenge (day 0). Data were analyzed using a slope basedweighted average approach. Open symbols denote measurements at LOD. Barsdenote medians.

FIG. 26. Survival (A) and % body weight change (B) of mice afterimmunization and challenge with H1N1 A/NL/602/09 as described in Example10.

FIG. 27. (A): Full length HA (HIJN A/Brisbane/59/2007) ELISA titers ofmice immunized as described in Example 10. Data were analyzed using aslope based weighted average approach. Open symbols denote measurementsat LOD. Bars denote medians. (B): Serum IgG CR9114 competition bindingobtained after immunization mice as described in Example 10. FL HA fromH1N1 A/Brisbane/59/2007 was used as the antigen. Data shown are groupmedians, error bars denote interquartile range. Data for CR9114 andCR8020 starting from a 5 μg/ml solution and diluted in the same manneras the serum samples are indicated.

FIG. 28. Primary screen of a total of 10472 clones (5544 and 4928 fromset 1 and 2, respectively) Data are normalized to the average of the FLHA binding and expression included in the experiment. The top 20%,clones in the CR9114 sandwich assay (panel A) also exhibitingexpression >50% of FL HA expression and binding signals to CR6261 >80%of the signals observed for FL HA (panel B) were considered hits; thisprocedure yielded 703 hits (596 and 107 from library 1 and 2,respectively).

FIG. 29. CR9114 sandwich Elisa results for polypeptides of the invention(A) SEQ ID NO: 158 to 162 all containing a C-terminal Flag-foldon-hissequence (B) SEQ ID NO: 163 to 166, all containing a C-terminal TCS-hissequence.

FIG. 30. SEC MALS results for SEQ ID NO: 158 in the presence and absenceof Fab fragments of CR9114 (indicated as CRF9114) or CR6261 (indicatedas CRF6261). The molecular mass derived from the multi-angle lightscattering analysis is given in example 12 and indicates formationcomplexes with 3 Fab fragments per trimer of the polypeptide of theinvention.

FIG. 31. Survival (A) and % body weight change (B) of mice afterimmunization and challenge with H1N1 A/Brisbane/59/07 as described inExample 13.

FIG. 32. (A): Full length HA (H1N1 A/Brisbane/59/2007) ELISA titers ofmice immunized as described in Example 13. Data were analyzed using aslope based weighted average approach. Open symbols denote measurementsat LOD. Bars denote medians. (B): Serum IgG CR9114 competition bindingobtained after immunization mice as described in Example 18. FL HA fromH1N1 A/Brisbane/59/2007 was used as the antigen. Data shown are groupmedians, error bars denote interquartile range. Levels for CR9114 andCR8020 starting from a 5 μg/ml solution and diluted in the same manneras the serum samples are indicated.

FIG. 33. Survival (A) and % body weight change (B) of mice afterimmunization and challenge with H5N1 A/Hon Kong/156/97 as described inExample 14.

FIG. 34. (A): Full length HA (H1N1 A/Brisbane/59/2007) ELISA titers ofmice immunized as described in Example 14. Data were analyzed using aslope based weighted average approach. Open symbols denote measurementsat LOD. Bars denote medians. (B): Serum IgG CR9114 competition bindingobtained after immunization mice as described in example 18. FL HA fromH1N1 A/Brisbane/59/2007 was used as the antigen. Data shown are groupmedians, error bars denote interquartile range. Levels for CR9114 andCR8020 starting from a 5 μg/ml solution and diluted in the same manneras the serum samples are indicated.

FIG. 35. Survival (A) and % body weight change (B) of mice afterimmunization and challenged with H1N A/Puerto Rico/8/1934 as describedin Example 15.

FIG. 36. (A): Full length HA (H1N1 A/Brisbane/59/2007) ELISA titers ofmice immunized as described in Example 15. Data were analyzed using aslope based weighted average approach. Open symbols denote measurementsat LOD. Bars denote medians. (B): Serum IgG CR9114 competition bindingobtained after immunization mice as described in example 18. FL HA fromH1N1 A/Brisbane/59/2007 was used as the antigen. Data shown are groupmedians, error bars denote interquartile range. Levels for CR9114 andCR8020 starting from a 5 μg/ml solution and diluted in the same manneras the serum samples are indicated.

DEFINITIONS

Definitions of terms as used in the present invention are given below.

An amino acid according to the invention can be any of the twentynaturally occurring (or ‘standard’ amino acids) or variants thereof,such as e.g. D-proline (the D-enantiomer of proline), or any variantsthat are not naturally found in proteins, such as e.g. norleucine. Thestandard amino acids can be divided into several groups based on theirproperties. Important factors are charge, hydrophilicity orhydrophobicity, size and functional groups. These properties areimportant for protein structure and protein-protein interactions. Someamino acids have special properties such as cysteine, that can formcovalent disulfide bonds (or disulfide bridges) to other cysteineresidues, proline that forms a cycle to the polypeptide backbone, andglycine that is more flexible than other amino acids. Table 2 shows theabbreviations and properties of the standard amino acids. The term“amino acid sequence identity” refers to the degree of identity orsimilarity between a pair of aligned amino acid sequences, usuallyexpressed as a percentage. Percent identity is the percentage of aminoacid residues in a candidate sequence that are identical (i.e., theamino acid residues at a given position in the alignment are the sameresidue) or similar (i.e., the amino acid substitution at a givenposition in the alignment is a conservative substitution, as discussedbelow), to the corresponding amino acid residue in the peptide afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence homology. Sequence homology, includingpercentages of sequence identity and similarity, are determined usingsequence alignment techniques well-known in the art, such as by visualinspection and mathematical calculation, or more preferably, thecomparison is done by comparing sequence information using a computerprogram. An exemplary, preferred computer program is the GeneticsComputer Group (GCG; Madison, Wis.) Wisconsin package version 10.0program, ‘GAP’ (Devereux et al. (1984)).

“Conservative substitution” refers to replacement of an amino acid ofone class is with another amino acid of the same class. In particularembodiments, a conservative substitution does not alter the structure orfunction, or both, of a polypeptide. Classes of amino acids for thepurposes of conservative substitution include hydrophobic (e.g. Met,Ala, Val, Leu), neutral hydrophilic (e.g. Cys, Ser, Thr), acidic (e.g.Asp, Glu), basic (e.g. Asn, Gin, His, Lys, Arg), conformation disrupters(e.g. Gly, Pro) and aromatic (e.g. Trp, Tyr, Phe).

As used herein, the terms “disease” and “disorder” are usedinterchangeably to refer to a condition in a subject. In someembodiments, the condition is a viral infection, in particular aninfluenza virus infection. In specific embodiments, a term “disease”refers to the pathological state resulting from the presence of thevirus in a cell or a subject, or by the invasion of a cell or subject bythe virus. In certain embodiments, the condition is a disease in asubject, the severity of which is decreased by inducing an immuneresponse in the subject through the administration of an immunogeniccomposition.

As used herein, the term “effective amount” in the context ofadministering a therapy to a subject refers to the amount of a therapywhich has a prophylactic and/or therapeutic effect(s). In certainembodiments, an “effective amount” in the context of administration of atherapy to a subject refers to the amount of a therapy which issufficient to achieve a reduction or amelioration of the severity of aninfluenza virus infection, disease or symptom associated therewith, suchas, but not limited to a reduction in the duration of an influenza virusinfection, disease or symptom associated therewith, the prevention ofthe progression of an influenza virus infection, disease or symptomassociated therewith, the prevention of the development or onset orrecurrence of an influenza virus infection, disease or symptomassociated therewith, the prevention or reduction of the spread of aninfluenza virus from one subject to another subject, the reduction ofhospitalization of a subject and/or hospitalization length, an increaseof the survival of a subject with an influenza virus infection ordisease associated therewith, elimination of an influenza virusinfection or disease associated therewith, inhibition or reduction ofinfluenza virus replication, reduction of influenza virus titer; and/orenhancement and/or improvement of the prophylactic or therapeuticeffect(s) of another therapy. In certain embodiments, the effectiveamount does not result in complete protection from an influenza virusdisease, but results in a lower titer or reduced number of influenzaviruses compared to an untreated subject. Benefits of a reduction in thetiter, number or total burden of influenza virus include, but are notlimited to, less severe symptoms of the infection, fewer symptoms of theinfection and a reduction in the length of the disease associated withthe infection.

The term “host”, as used herein, is intended to refer to an organism ora cell into which a vector such as a cloning vector or an expressionvector has been introduced. The organism or cell can be prokaryotic oreukaryotic. Preferably, the host comprises isolated host cells, e.g.host cells in culture. The term “host cells” merely signifies that thecells are modified for the (over)-expression of the polypeptides of theinvention. It should be understood that the term host is intended torefer not only to the particular subject organism or cell but to theprogeny of such an organism or cell as well. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent organism or cell, but are still included within the scopeof the term “host” as used herein.

The term “included” or “including” as used herein is deemed to befollowed by the words “without limitation”.

As used herein, the term “infection” means the invasion by,multiplication and/or presence of a virus in a cell or a subject. In oneembodiment, an infection is an “active” infection, i.e., one in whichthe virus is replicating in a cell or a subject. Such an infection ischaracterized by the spread of the virus to other cells, tissues, and/ororgans, from the cells, tissues, and/or organs initially infected by thevirus. An infection may also be a latent infection, i.e., one in whichthe virus is not replicating. In certain embodiments, an infectionrefers to the pathological state resulting from the presence of thevirus in a cell or a subject, or by the invasion of a cell or subject bythe virus.

Influenza viruses are classified into influenza virus types: genus A, Band C. The term “influenza virus subtype” as used herein refers toinfluenza A virus variants that are characterized by combinations of thehemagglutinin (H) and neuramidase (N) viral surface proteins. Accordingto the present invention influenza virus subtypes may be referred to bytheir H number, such as for example “influenza virus comprising HA ofthe H3 subtype”, “influenza virus of the H3 subtype” or “H3 influenza”,or by a combination of a H number and an N number, such as for example“influenza virus subtype H3N2” or “H3N2”. The term “subtype”specifically includes all individual “strains”, within each subtype,which usually result from mutations and show different pathogenicprofiles, including natural isolates as well as man-made mutants orreassortants and the like. Such strains may also be referred to asvarious “isolates” of a viral subtype. Accordingly, as used herein, theterms “strains” and “isolates” may be used interchangeably. The currentnomenclature for human influenza virus strains or isolates includes thetype (genus) of virus, i.e. A, B or C, the geographical location of thefirst isolation, strain number and year of isolation, usually with theantigenic description of HA and NA given in brackets, e.g.A/Moscow/10/00 (H3N2). Non-human strains also include the host of originin the nomenclature. The influenza A virus subtypes can further beclassified by reference to their phylogenetic group. Phylogeneticanalysis has demonstrated a subdivision of hemagglutinins into two maingroups: inter alia the H1, H2, H5 and H9 subtypes in phylogenetic group1 (“group 1” influenza viruses) and inter alia the H3, H4, H7 and H10subtypes in phylogenetic group 2 (“group 2” influenza viruses).

As used herein, the term “influenza virus disease” refers to thepathological state resulting from the presence of an influenza virus,e.g. an influenza A or B virus in a cell or subject or the invasion of acell or subject by an influenza virus. In specific embodiments, the termrefers to a respiratory illness caused by an influenza virus.

As used herein, the term “nucleic acid” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid can be single-stranded or double-stranded. The nucleic acidmolecules may be modified chemically or biochemically or may containnon-natural or derivatized nucleotide bases, as will be readilyappreciated by those of skill in the art. Such modifications include,for example, labels, methylation, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). A reference to a nucleic acid sequence encompassesits complement unless otherwise specified. Thus, a reference to anucleic acid molecule having a particular sequence should be understoodto encompass its complementary strand, with its complementary sequence.The complementary strand is also useful, e.g., for anti-sense therapy,hybridization probes and PCR primers.

As used herein, in certain embodiments the numbering of the amino acidsin HA is based on the numbering of amino acids in HA0 of a wild typeinfluenza virus, e.g. the numbering of the amino acids of the H1N1influenza strain A/Brisbane/59/2007 (SEQ ID NO: 1). As used in thepresent invention, the wording “the amino acid at position “x” in HA”thus means the amino acid corresponding to the amino acid at position xin HA0 of the particular wild type influenza virus, e.g.A/Brisbane/59/2007 (SEQ ID NO: 1; wherein the amino acids of the HA2domain have been indicated in italics). It will be understood by theskilled person that equivalent amino acids in other influenza virusstrains and/or subtypes can be determined by multiple sequencealignment. Note that, in the numbering system used throughout thisapplication 1 refers to the N-terminal amino acid of an immature HA0protein (SEQ ID NO: 1). The mature sequence starts e.g. on position 18of SEQ ID NO: 1. It will be understood by the skilled person that theleader sequence (or signal sequence) that directs transport of a proteinduring production (e.g. corresponding to amino acids 1-17 of SEQ ID NO:1), generally is not present in the final polypeptide, that is e.g. usedin a vaccine. In certain embodiments, the polypeptides according to theinvention thus comprise an amino acid sequence without the leadersequence, i.e. the amino acid sequence is based on the amino acidsequence of HA0 without the signal sequence.

“Polypeptide” refers to a polymer of amino acids linked by amide bondsas is known to those of skill in the art. As used herein, the term canrefer to a single polypeptide chain linked by covalent amide bonds. Theterm can also refer to multiple polypeptide chains associated bynon-covalent interactions such as ionic contacts, hydrogen bonds, Vander Waals contacts and hydrophobic contacts. Those of skill in the artwill recognize that the term includes polypeptides that have beenmodified, for example by post-translational processing such as signalpeptide cleavage, disulfide bond formation, glycosylation (e.g.,N-linked and O-linked glycosylation), protease cleavage and lipidmodification (e.g. S-palmitoylation).

“Stem domain polypeptide” refers to a polypeptide that comprises one ormore polypeptide chains that make up a stem domain of anaturally-occurring (or wild-type) hemagglutinin (HA). Typically, a stemdomain polypeptide is a single polypeptide chain (i.e. corresponding tothe stem domain of a hemagglutinin HA0 polypeptide) or two polypeptidechains (i.e. corresponding to the stem domain of a hemagglutinin HA1polypeptide in association with a hemagglutinin HA2 polypeptide).According to the invention, a stem domain polypeptide comprises one ormore mutations as compared to the wild-type HA molecule, in particularone or more amino acid residues of the wild-type HA may have beensubstituted by other amino acids, not naturally occurring on thecorresponding position in a particular wild-type HA. Stem domainpolypeptides according to the invention can furthermore comprise one ormore linking sequences, as described below.

The term “vector” denotes a nucleic acid molecule into which a secondnucleic acid molecule can be inserted for introduction into a host whereit will be replicated, and in some cases expressed. In other words, avector is capable of transporting a nucleic acid molecule to which ithas been linked. Cloning as well as expression vectors are contemplatedby the term “vector”, as used herein. Vectors include, but are notlimited to, plasmids, cosmids, bacterial artificial chromosomes (BAC)and yeast artificial chromosomes (YAC) and vectors derived frombacteriophages or plant or animal (including human) viruses. Vectorscomprise an origin of replication recognized by the proposed host and incase of expression vectors, promoter and other regulatory regionsrecognized by the host. Certain vectors are capable of autonomousreplication in a host into which they are introduced (e.g., vectorshaving a bacterial origin of replication can replicate in bacteria).Other vectors can be integrated into the genome of a host uponintroduction into the host, and thereby are replicated along with thehost genome. As used herein, the term “wild-type” in the context of avirus refers to influenza viruses that are prevalent, circulatingnaturally and producing typical outbreaks of disease.

DETAILED DESCRIPTION

Influenza viruses have a significant impact on global public health,causing millions of cases of severe illness each year, thousands ofdeaths, and considerable economic losses. Current trivalent influenzavaccines elicit a potent neutralizing antibody response to the vaccinestrains and closely related isolates, but rarely extend to more divergedstrains within a subtype or to other subtypes. In addition, selection ofthe appropriate vaccine strains presents many challenges and frequentlyresults in sub-optimal protection. Furthermore, predicting the subtypeof the next pandemic virus, including when and where it will arise, iscurrently impossible.

Hemagglutinin (HA) is the major envelope glycoprotein from influenza Aviruses which is the major target of neutralizing antibodies.Hemagglutinin has two main functions during the entry process. First,hemagglutinin mediates attachment of the virus to the surface of targetcells through interactions with sialic acid receptors. Second, afterendocytosis of the virus, hemagglutinin subsequently triggers the fusionof the viral and endosomal membranes to release its genome into thecytoplasm of the target cell. HA comprises a large ectodomain of ˜500amino acids that is cleaved by host-derived enzymes to generate 2polypeptides that remain linked by a disulfide bond. The majority of theN-terminal fragment (HA1, 320-330 amino acids) forms a membrane-distalglobular domain that contains the receptor-binding site and mostdeterminants recognized by virus-neutralizing antibodies. The smallerC-terminal portion (HA2, ˜180 amino acids) forms a stem-like structurethat anchors the globular domain to the cellular or viral membrane. Thedegree of sequence homology between subtypes is smaller in the HA1polypeptides (34%-59% homology between subtypes) than in the HA2polypeptide (51%-80% homology). The most conserved region is thesequence around the cleavage site, particularly the HA2 N-terminal 23amino acids, which is conserved among all influenza A virus subtypes(Lorieau et al., 2010). Part of this region is exposed as a surface loopin the HA precursor molecule (HA0), but becomes inaccessible when HA0 iscleaved into HA1 and HA2.

Most neutralizing antibodies bind to the loops that surround thereceptor binding site and interfere with receptor binding andattachment. Since these loops are highly variable, most antibodiestargeting these regions are strain-specific, explaining why currentvaccines elicit such limited, strain-specific immunity. Recently,however, fully human monoclonal antibodies against influenza virushemagglutinin with broad cross-neutralizing potency were generated.Functional and structural analysis have revealed that these antibodiesinterfere with the membrane fusion process and are directed againsthighly conserved epitopes in the stem domain of the influenza HA protein(Throsby et al., 2008; Ekiert et al. 2009, WO 2008/028946,WO2010/130636, WO 2013/007770).

Stem domain polypeptides stably presenting the epitopes of theseantibodies are described in the co-pending patent applicationPCT/EP2012/073706. At least some of the stem domain polypeptidesdescribed herein stably present the epitope of CR6261 and/or CR9114 andare immunogenic in mice. Additional immunogenic stem domain polypeptidesstably presenting the epitope of CR6261 and/or CR9114 have beendescribed in co-pending patent application PCT/EP2014/060997.

According to the present invention new HA stem domain polypeptides havebeen designed presenting these epitopes. These polypeptides can be usedto create a universal epitope-based vaccine inducing protection againsta broad range of influenza strains. Like in the previously describedstem domain polypeptides, the highly variable and immunodominant part,i.e. the head domain, is first removed from the full length HA moleculeto create a stem domain polypeptide, also called mini-HA, in order toredirect the immune response towards the stem domain where the epitopesfor the broadly neutralizing antibodies are located. The broadlyneutralizing antibodies mentioned above were used to probe the correctfolding of the newly created molecules, and to confirm the presence ofthe neutralizing epitopes.

The new stem domain polypeptides of the invention show increased bindingof the antibodies, in particular CR6261 and/or CR9114, and/or anincreased propensity to multimerize and increased stability, as comparedto binding of those antibodies to the stem polypeptides describedearlier (PCT/EP2012/073706 and PCT/EP2014/060997).

The stem domain polypeptides of this invention are capable of presentingthe conserved epitopes of the membrane proximal stem domain HA moleculeto the immune system in the absence of dominant epitopes that arepresent in the membrane distal head domain. To this end, part of theprimary sequence of the HA0 protein making up the head domain is removedand reconnected, either directly or, in some embodiments, by introducinga short flexible linking sequence (‘linker’) to restore the continuityof the polypeptide chain. The resulting polypeptide sequence is furthermodified by introducing specific mutations that stabilize the native3-dimensional structure of the remaining part of the HA0 molecule.

The present invention in particular provides influenza hemagglutininstem domain polypeptides comprising:

-   -   (a) an influenza hemagglutinin HA1 domain that comprises an HA1        N-terminal stem segment, covalently linked by a linking sequence        of 0-50 amino acid residues to an HA1 C-terminal stem segment,        said HA1 C-terminal segment being linked to    -   (b) an influenza hemagglutinin HA2 domain, wherein the HA1 and        HA2 domain are derived from an influenza A virus subtype        comprising HA of the H1 subtype;    -   (c) wherein the polypeptide comprises no protease cleavage site        at the junction between the HA1 and HA2 domain;    -   (d) wherein said HA1 N-terminal segment comprises the amino        acids 1-x of HA1, preferably the amino acids p-x of HA1, and        wherein the HA1 C-terminal stem segment comprises the amino        acids y-C-terminal amino acid of HA1, wherein x=the amino acid        on position 52 of SEQ ID NO: 1 (or an equivalent position in        hemagglutinin of another influenza virus strain), p=the amino        acid on position 18 of SEQ ID NO: 1 (or an equivalent position        in hemagglutinin of another influenza virus) and y=the amino        acid on position 321 of SEQ ID NO: 1 (or an equivalent position        in another hemagglutinin);    -   (e) wherein the region comprising the amino acid residues        402-418 comprises the amino acid sequence        X₁NTQX₂TAX₃GKEX₄N(H/K)X₅E(K/R) (SEQ ID NO: 8), wherein:    -   X₁, is an amino acid selected from the group consisting of M, E,        K, V, R and T.    -   X₂ is an amino acid selected from the group consisting of F, I,        N, T, H, L and Y, preferably I, L or Y,    -   X₃ is an amino acid selected from the group consisting of V, A,        G, I, R, F and S, preferably A, I or F,    -   X₄, is an amino acid selected from the group consisting of F, I,        N, S, T, Y, E, K, M, and V, preferably I, Y, M or V,    -   X₅ is an amino acid selected from the group consisting of L, H,        I, N, R, preferably I;    -   (f) wherein the amino acid residue on position 337 (HA1 domain)        is selected from the group consisting of: I, E, K, V, A, and T,    -   the amino acid residue on position 340 (HA1 domain) is selected        from the group consisting of: I, K, R, T, F, N, S and Y,    -   the amino acid residue on position 352 (HA2 domain) is selected        from the group consisting of: D, V, Y, A, I, N, S, and T, and    -   the amino acid residue on position 353 (HA2 domain) is selected        from the group consisting of: K, R, T, E, G, and V; and    -   (g) wherein the polypeptide further comprises a disulfide bridge        between the amino acid on position 324 and the amino acid on        position 436; and    -   (h) wherein the amino acid sequence RMKQIEDKIEEIESK (SEQ ID        NO: 20) has been introduced at positions 419-433 or wherein        sequence RMKQIEDKIEEIESKQK (SEQ ID NO: 21) has been introduced        at position 417-433.

The present invention thus provides stable hemagglutinin stempolypeptides that mimic the three-dimensional conformation of the stemof the natural hemagglutinin molecule.

The polypeptides of the invention do not comprise the full length HA1domain.

The polypeptides thus are substantially smaller than HA0, preferablylacking all or substantially all of the globular head of HA. Preferably,the immunogenic polypeptides are no more than 360, preferably no morethan 350, 340, 330, 320, 310, 305, 300, 295, 290, 285, 280, 275, or 270amino acids in length. In certain embodiments, the immunogenicpolypeptides are from about 250 to about 350, preferably from about 260to about 340, preferably from about 270 to about 330, preferably fromabout 270 to about 330 amino acids in length.

According to the invention, the “HA1 N-terminal segment” refers to apolypeptide segment that corresponds to the amino-terminal portion ofthe HA1 domain of an influenza hemagglutinin (HA) molecule. The HA1N-terminal polypeptide segment comprises the amino acids from position 1to position x of the HA1 domain, wherein the amino acid on position x isan amino acid residue within HA1. The term “HA1 C-terminal segment”refers to a polypeptide segment that corresponds to the carboxy-terminalportion of an influenza hemagglutinin HA1 domain. The HA1 C-terminalpolypeptide segment comprises the amino acids from position y to andincluding the C-terminal amino acid of the HA1 domain, wherein the aminoacid on position y is an amino acid residue within HA1. According to theinvention y is greater than x, thus a segment of the HA1 domain betweenthe HA1 N-terminal segment and the HA1 C-terminal segment, i.e. betweenthe amino acid on position x and the amino acid on position y of HA1,has been deleted, and in some embodiments, replaced by a linkingsequence. Thus, in certain embodiments, the deletion in the HA1 segmentcomprises the amino acid sequence from the amino acid at position x+1 upto and including the amino acid at position y-1.

In certain embodiments, the polypeptides do not comprise the signalsequence. Thus in certain embodiments, the HA1 N-terminal segmentcomprises the amino acid p-x of HA1, wherein p is the first amino acidof the mature HA molecule (e.g. p=18 in case of SEQ ID NO: 1). Theskilled person will be able to determine the equivalent amino acid inother hemagglutins and to prepare the polypeptides described hereinwithout the signal peptides (e.g. amino acids 1-17 of SEQ ID NO: 1 or anequivalent position in other H1 influenza virus strains (see e.g. Table2), to position x of the HA1 domain.

According to the present invention, the HA1 N-terminal segment comprisesthe amino acids 1-x, preferably p-x of the HA1 domain, wherein x=52 andp=18 in SEQ ID NO: 1 or an equivalent amino acid position in other HAsequences of the H1 subtype.

According to the invention, the HA1 C-terminal polypeptide segmentcomprises the amino acids from position y to and including theC-terminal amino acid of the H1 HA1 domain, wherein y is 321 or anequivalent amino acid position in other HA sequences of the H1 subtype.

According to the invention, the HA1 N-terminal stem segment thuscomprises the amino acid residues 1-52 of HA1, preferably the amino acidresidues 18-52 of HA1, and the HA1 C-terminal stem segment comprises theamino acid residues 321-343 of HA1. In certain embodiments, the HA1N-terminal stem segment consists of the amino acid residues 1-52 of HA1,preferably the amino acid residues 18-52 of HA1, and the HA1 C-terminalstem segment consists of the amino acid residues 321-343 of HA1.

According to the invention, the polypeptides do not comprise a proteasecleavage site at the junction between the HA1 and the HA2 domain. Thus,the hemagglutinin stem domain polypeptides are resistant to proteasecleavage at the junction between HA1 and HA2. It is known to those ofskill in the art that the Arg (R)-Gly (G) sequence spanning HA1 and HA2(i.e. amino acid positions 343 and 344 in SEQ ID NO: 1) is a recognitionsite for trypsin and trypsin-like proteases and is typically cleaved forhemagglutinin activation. Since the HA stem domain polypeptidesdescribed herein should not be activated, the influenza hemagglutininstem domain polypeptides of the invention are resistant to proteasecleavage. According to the invention, the protease cleavage site thushas been removed in order to prevent cleavage of the polypeptide at thecleavage site between the HA1 and HA2 domain. In certain embodiments,the protease cleavage site has been removed by mutation of theC-terminal amino acid of the C-terminal HA1 segment and/or mutation ofthe N-terminal amino acid of the HA2 domain to obtain a sequence that isresistant to protease cleavage. In certain embodiments, removal of thecleavage site between HA1 and HA2 in certain embodiments can be achievedby mutation of R (in a small number of cases K) to Q at the P1 position(see e.g. Sun et al, 2010 for an explanation of the nomenclature of thecleavage site (position 343 in SEQ ID NO: 1). Thus, in certainembodiments, the C-terminal amino acid residue of the HA1 C-terminalstem segment is any amino acid other than arginine (R) or lysine (K). Incertain embodiments, the HA1 C-terminal amino acid is glutamine (Q),serine (S), threonine (T), asparagine (N), aspartic acid (D) or glutamicacid (E). In certain embodiments, the C-terminal amino acid residue ofthe HA1 C-terminal stem segment is glutamine (Q).

According to the invention, the polypeptides are derived from or basedon H1 HA, i.e. HA comprising an amino acid sequence from an influenzavirus of the H1 subtype. In a particular embodiment, the polypeptidescomprise hemagglutinin stem domains from or based on HA of an influenzaA virus comprising HA of the H1 subtype, such as from the influenzavirus A/Brisbane/59/2007 (H1N1) (SEQ ID NO:1), as described below. Itwill be understood by the skilled person that also other influenza Aviruses comprising HA of the H1 subtype may be used according to theinvention. In certain embodiments, the polypeptides comprisehemagglutinin stem domains derived from or based on HA of an influenza AH1 virus selected from Table 2. With “derived from” or “based on” it ismeant that the N-terminal segments, and/or C-terminal segments of theHA1 domain and/or the HA2 domains have at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% amino acid sequence identity with thecorresponding N-terminal and/or C-terminal segments of HA1 and/or theHA2 domains of a naturally occurring influenza hemagglutinin of a H1subtype known to those of skill in the art or later discovered.

According to the invention, the HA2 domain comprises one or moremutations in the HA2 amino acid sequence connecting the C-terminalresidue of helix A to the N-terminal residue of helix CD (FIG. 1). TheH1 HA2 amino acid sequence connecting the C-terminal residue of helix Aand the N-terminal residue of helix CD comprises the amino acid sequencecomprising residues 402-418 of influenza HA (numbering according to SEQID NO: 1), comprising the amino acid sequence MNTQFTAVGKEFN(H/K)LE(K/R)(SEQ ID NO: 7).

In certain embodiments, the amino acid sequence connecting theC-terminal residue of helix A to the N-terminal residue of helix CD,i.e. the region comprising the amino acid residues 402-418 of influenzaHA of serotype H1 (numbering according to SEQ ID NO: 1) comprises theamino acid sequence X₁NTQX₂TAX₃GKEX₄N(H/K)X₅E(K/R) (SEQ ID NO: 8).

According to the invention, one or more of the amino acids on position402, 406, 409, 413 and 416 (numbering refers to SEQ ID NO: 1), i.e oneor more of the amino acids X₁, X₂, X₃, X₄ and X, have been mutated, i.e.comprise an amino acid that is not occurring at those positions in awild-type influenza virus on which the stem polypeptide is based.

In certain embodiments, the mutated amino acid on position 402, i.e. X₁is an amino acid selected from the group consisting of M, E, K, V, R andT.

In certain embodiments, the mutated amino acid on position 406, i.e. X₂is an amino acid selected from the group consisting of F, I, N, T, H, Land Y, preferably I, L or Y.

In certain embodiments, the mutated amino acid on position 409, i.e. X₃,is an amino acid selected from the group consisting of V, A, G, I, R, Fand S, preferably A, I or F.

In certain embodiments, the mutated amino acid on position 413, i.e. X₄,is an amino acid selected from the group consisting of F, I, N, S, T, Y,E, K, M, and V, preferably I, Y, M or V.

In certain embodiments, the mutated amino acid on position 416, i.e. X₅is an amino acid selected from the group consisting of L, H, I, N, R,preferably I. Combinations of these mutations are also possible.

In certain embodiments, X₁ is M, X₂ is Y, X₃ is 1, X₄ is Y and X₅ is S.

According to the invention, the stem polypeptides comprise one or moreadditional mutations, i.e. amino acid substitutions, in the HA1 domainand/or the HA2 domain, as compared to the amino acid sequence ofcorresponding wild-type influenza virus HA1 and/or HA2 domains, i.e. theinfluenza virus on which the stem polypeptides are based.

In certain embodiments, one or more amino acid residues close to the HA0cleavage site (residue 343 in SEQ ID NO: 1) have been mutated. Incertain embodiments, one or more of the amino acid residues on position337, 340, 352, or 353 of SEQ ID NO: 1, or equivalent positions in otherinfluenza viruses, have been mutated, i.e. are substituted by an aminoacid that is not occurring at the corresponding position in the aminoacid sequence of the HA of the wild-type influenza virus on which thestem polypeptide is based. Table 6 shows the the naturally occurringamino acid variation.

In certain embodiments, the polypeptides of the invention comprise atleast one mutation on position 352 of SEQ ID NO: 1, or on an equivalentposition of other influenza viruses.

In certain embodiments, the polypeptides of the invention comprise atleast one mutation on position 353 of SEQ ID NO: 1, or on an equivalentposition of other influenza viruses.

In certain embodiments, the polypeptides of the invention comprise atleast one mutation on position 337 of SEQ ID NO: 1, or on an equivalentposition of other influenza viruses.

In certain embodiments, the polypeptides of the invention comprise atleast one mutation on position 340 of SEQ ID NO: 1, or on an equivalentposition of other influenza viruses.

In certain embodiments, the mutated amino acid residue on position 337(HA1 domain) is selected from the group consisting of: I, E, K, V, A,and T.

In certain embodiments, the mutated amino acid residue on position 340(HA1 domain) is selected from the group consisting of: I, K, R, T, F, N,S and Y.

In certain embodiments, the mutated amino acid residue on position 352(HA2 domain) is selected from the group consisting of: D, V, Y, A, I, N,S, and T.

In certain embodiments, the mutated amino acid residue on position 353(HA2 domain) is selected from the group consisting of: K, R, T, E, G,and V.

In certain embodiments the mutated amino acid introduces a consensusN-glycoslation e.g. N-X-T/S (where X is any naturally curing amino acidexcept P) in the sequence as is for example the case for I340N in SEQ IDNO: 6.

In certain embodiments, the mutated amino acid is an amino acid thatdoes not naturally occur in sequences of the same subtype.

In certain embodiments, the the mutated amino acid residue on position337 (HA1 domain) is K.

In certain embodiments, the mutated amino acid residue on position 340(HA1 domain) is K.

In certain embodiments, the mutated amino acid residue on position 352(HA2 domain) is F.

In certain embodiments, the mutated amino acid residue on position 353(HA2 domain) is T.

It is again noted that throughout this application the numbering of theamino acids is based on the numbering of amino acids in H1 HA0, inparticular the numbering of the amino acids of the H1N1 influenza strainA/Brisbane/59/2007 (SEQ ID NO: 1). The skilled person will be able todetermine the equivalent (or corresponding) amino acids in HA of otherinfluenza viruses and thus will be able to determine equivalentmutations, see e.g. Table 2 for the sequence alignment of different H1influenza viruses. According to the invention, the polypeptides furthercomprise a disulfide bridge between the amino acid on position 324 andthe amino acid on position 436. Thus, according to the invention atleast one disulfide bridge has been introduced in the stem domainpolypeptides, preferably between amino acids of (or the equivalent of)position 324 and 436 in H1 A/Brisbane/59/2007 (SEQ ID NO: 1). In certainembodiments, the polypeptides thus further comprise the mutation R324Cin the HA1 domain and T436C in the HA2 domain. Equivalent positions canbe easily determined by those skilled in the art by aligning thesequences using a suitable algorithm such as Clustal, Muscle etc.Engineered disulfide bridges are created by mutating at least one (ifthe other is already a cysteine), but usually two residues that arespatially close into cysteine, that will spontaneously or by activeoxidation form a covalent bond between the sulfur atoms of theseresidues.

In certain embodiments, the polypeptides further comprise one or moreadditional mutations in the HA1 and/or HA2 domain, as compared to theamino acid sequence of the HA of which the HA1 and HA2 domains arederived. Thus, the stability of the stem polypeptides is furtherincreased.

Applicants have previously identified broadly neutralizing antibodiesisolated from primary human B-cells from vaccinated individuals some ofwhich were specific for group 1 (e.g. CR6261, as described in WO2008/028946) and some of which were specific for group 2 influenzaviruses (e.g. CR8020 as described in WO 2010/130636). Detailed analysisof the epitopes of these monoclonal antibodies has revealed the reasonfor the lack of cross-reactivity of these specific antibodies. In bothcases the presence of glycans in group 1 or group 2 HA molecules ondifferent positions at least partly explained the fact that theantibodies are group-specific. With the identification of CR9114-likeantibodies that cross-react with many group 1 and 2 HA molecules, asdescribed below, it has become clear that it is possible for the humanimmune system to elicit very broad neutralizing antibodies againstinfluenza viruses. However, given the need for a yearly vaccinationscheme these antibodies are apparently not, or only to a very low extentelicited following infection or vaccination with (seasonal) influenzaviruses of subtypes H1 and/or H3.

According to the present invention polypeptides are provided that mimicthe specific epitopes of CR6261 and/or CR9114, and that can be used asimmunogenic polypeptides, e.g. to elicit cross-neutralizing antibodieswhen administered in vivo, either alone, or in combination with otherprophylactic and/or therapeutic treatments. With “cross-neutralizingantibodies”, antibodies are meant that are capable of neutralizing atleast two, preferably at least three, four, or five different subtypesof influenza A viruses of phylogenetic group 1, and/or at least two,preferably at least three, four, or five different subtypes of influenzaA viruses of phylogenetic group 2, and/or at least two, differentsubtypes of influenza B viruses, in particular at least all virusstrains that are neutralized by CR6261 and CR9114.

The polypeptides of the invention comprise the epitope of thestem-binding influenza neutralizing antibodies CR6261 and/or CR9114. Incertain embodiments, the polypeptides thus selectively bind to theantibodies CR6261 and/or CR9114. In certain embodiments, thepolypeptides of the invention do not bind to the antibodies CR8020and/or CR8057. As used in the present invention, CR6261 comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 9 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 10; CR9114 comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 11 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:12. CR8057 comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 13 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 14. CR8020 comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 17 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 18.

As described above, the polypeptides comprise an influenza hemagglutininHA1 domain that comprises an HA1 N-terminal stem segment that iscovalently linked by a linking sequence of 0-50 amino acid residues tothe HA1 C-terminal stem segment. The linking sequence, if present, doesnot occur in naturally occurring, or wild-type, HA. In certainembodiments, the linker is a peptide that comprises one amino acidresidue, two or less amino acid residues, three or less amino acidresidues, four or less amino acid residues, five or less amino acidresidues, ten or less amino acid residues, 15 or less amino acidresidues, or 20 or less amino acid residues or 30 or less amino acidresidues or or less amino acid residues or 50 or less amino acidresidues. In a specific embodiment, the linking sequence is a sequenceselected from the group consisting of G, GS, GGG, GSG, GSA, GSGS, GSAG,GGGG, GSAGS, GSGSG, GSAGSA, GSAGSAG, and GSGSGSG.

In certain embodiments, the HA1 N-terminal segment is directly linked tothe HA1 C-terminal segment, i.e. the polypeptides do not comprise alinking sequence.

Influenza HA in its native form exists as a trimer on the cell or virusmembrane. In certain embodiments the intracellular and transmembranesequence is removed so that a secreted (soluble) polypeptide is producedfollowing expression in cells. Methods to express and purify secretedectodomains of HA have been described (see e.g. Dopheide et al 2009;Ekiert et al 2009, 2011; Stevens et al 2004, 2006; Wilson et al 1981). Aperson skilled in the art will understand that these methods can also beapplied directly to stem domain polypeptides of the invention in orderto achieve expression of secreted (soluble) polypeptide. Therefore thesepolypeptides are also encompassed in the invention.

In certain embodiments, the polypeptides comprise the full HA2 domain,thus including the transmembrane and intracellular sequences. In otherembodiments, the polypeptides of the invention do not comprise theintracellular sequences of HA and the transmembrane domain. In certainembodiments, the polypeptides comprise a truncated HA2 domain. Incertain embodiments, the intracellular and transmembrane sequences, e.g.the amino acid sequence from position (or the equivalent of) 514, 515,516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 526, 528,529, or 530 of the HA2 domain to the C-terminus of the HA2 domain(numbering according to SEQ ID NO: 1) has been removed to produce asoluble polypeptide following expression in cells.

In certain embodiments, the C-terminal part of the HA2 domain fromposition 519 to the C-terminal amino acid has been deleted. In furtherembodiments, the C-terminal part of the HA2 domain from position 530 tothe C-terminal amino acid has been deleted.

Optionally, a his-tag sequence (HHHHHH (SEQ ID NO: 15) or HHHHHHH (SEQID NO: 16)) may be linked to the (optionally truncated) HA2 domain, forpurification purposes, optionally connected through a linker. Optionallythe linker may contain a proteolytic cleavage site to enzymaticallyremove the his-tag after purification.

In certain embodiments, the polypeptides are further stabilized byintroducing a sequence known to form trimeric structures, i.e.GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 3) at the C-terminus of HA2,optionally connected through a linker. Thus, in certain embodiments, theC-terminal part of the HA2 domain has been replaced by the amino acidsequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 3), optionallyconnected through a linker. The linker may optionally contain a cleavagesite for processing afterwards according to protocols well known tothose skilled in the art. To facilitate purification of the soluble forma tag sequence may be added, e.g. a his tag (HHHHHH (SEQ ID NO: 15) orHHHHHHH (SEQ ID NO: 16)) or FLAG tag (DYKDDDDK) (SEQ ID NO: 22) or acombination of these, optionally connected via short linkers. The linkermay optionally contain (part of) a proteolytic cleavage site, e.g. IEGR(SEQ ID NO: 24) (Factor X) or LVPRGS (SEQ ID NO: 23) (thrombin) forprocessing afterwards according to protocols well known to those skilledin the art. The processed proteins are also encompassed in theinvention.

In certain embodiments, the C-terminal part of the HA2 domain fromposition 519-565 has been deleted (numbering according to SEQ ID NO: 1)and replaced by SGRDYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHH H(SEQ ID NO: 4).

In certain embodiments, the C-terminal part of the HA2 domain fromposition 530-565 has been deleted (numbering according to SEQ ID NO: 1)and replaced by SGRDYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHH H(SEQ ID NO: 4).

The native HA exists as a trimer on the cell surface. Most of theinteractions between the individual monomers that keep the trimertogether are located in the head domain while in the stem domaintrimerization is mediated by the formation of a trimeric coiled coilmotif. After removal of the head the tertiary structure is destabilizedand therefore modifications are needed in order to increase proteinstability. By strengthening the helical propensity of the helix CD amore stable protein can be created. In the polypeptides described in theco-pending application PCT/EP2014/060997, the sequence MKQIEDKIEEIESKQ(SEQ ID NO: 5), derived from yeast transcriptional activator proteinGCN4 and known to trimerize was introduced in the CD helix at (theequivalent of) position 419-433. This sequence has a high propensity toform helical secondary structures and can enhance in this way overallstability of the polypeptides of the invention.

According to the present invention, it has surprisingly been shown thatthe stability and multimerization state of the polypeptide is dependenton the exact location and sequence of the GCN4 derived sequence in theprimary sequence of the polypeptides of the invention.

Thus, according the invention, the sequence RMKQIEDKIEEIESK (SEQ ID NO:20) is introduced at position 419-433 (numbering according to SEQ ID NO:I), or sequence RMKQIEDKIEEIESKQK (SEQ ID NO: 21) is introduced atposition 417-433. In certain embodiments, the polypeptides areglycosylated.

In the research that led to the present invention, for example s74H9(SEQ ID NO: 65), s127H1 (SEQ ID NO: 66), s71H2 (SEQ ID NO: 71), s86B4(SEQ ID NO: 67), s115A1 (SEQ ID NO: 70), s2201C9 (SEQ ID NO: 77), s55G7(SEQ ID NO: 68), s113E7 (SEQ ID NO: 78), s6E12 (SEQ ID NO: 69), s181H9(SEQ ID NO: 76), described in the co-pending patent applicationPCT/EP2014/060997 were modified, using techniques of molecular biologywell known to those skilled in the art, to create sequences s74H9-t2(SEQ ID NO: 93), s127H1-t2 (SEQ ID NO: 91), s71H2-t2 (SEQ ID NO: 97),s86B4-t2 (SEQ ID NO: 92), s115A1-t2 (SEQ ID NO: 96), s220C9-t2 (SEQ IDNO: 99), s55G7-t2 (SEQ ID NO: 95), s113E7-t2 (SEQ ID NO: 100), s6E12-t2(SEQ ID NO: 94), s181H9-t2 (SEQ ID NO: 98) containing sequenceRMKQIEDKIEEIESK (SEQ ID NO: 20) at position 419-433. In a similarmanner, polypeptides s74H9-t3 (SEQ ID NO: 123), s127H1-t3 (SEQ ID NO:121), s71H2-t3 (SEQ ID NO: 127), s86B4-t3 (SEQ ID NO: 122), s115A1-t3(SEQ ID NO: 126), s2201C9-t3 (SEQ ID NO: 129), s55G7-t3 (SEQ ID NO:125), s113E7-t3 (SEQ ID NO: 130), s6E12-t3 (SEQ ID NO: 124), s181H9-t3(SEQ ID NO: 128) containing sequence RMKQIEDKIEEIESKQK (SEQ ID NO: 21)at position 417-433 were created.

The polypeptides of the present invention show increased binding of theinfluenza antibodies, in particular CR6261 and/or CR9114, and/or anincreased propensity to multimerize and/or an increased stability, ascompared to stem polypeptides described earlier (PCT/EP2012/073706 andPCT/EP2014/060997).

In certain embodiments, the polypeptides comprise the amino acidsequence:

(SEQ ID NO: 145) DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNX ₁PSX ₂QSQGLFGAIAGX ₃ X ₄EGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKX ₅NTQX ₆TAX ₇GKEX ₈NKX ₉ERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVSGRDYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH,wherein X₁ is an amino acid selected from the group consisting of E, I,K, V, A, and T;X₂ is an amino acid selected from the group consisting of I, K, R, T, F,N, S and Y;X₃ is an amino acid selected from the group consisting of D, F, V, Y, A,I, N, S, and T;X₄ is an amino acid selected from the group consisting of I, K, R, T, E,G and V;X₅ is an amino acid selected from the group consisting of M, E, K, V, R,T;X₆ is an amino acid selected from the group consisting of F, I, N, S, T,Y, H, and L;X₇ is an amino acid selected from the group consisting of A, G, I, R, T,V, F, and S;X₈ is an amino acid selected from the group consisting of F, I, N, S, T,Y, G, E, K, M, and V; andX₉ is an amino acid selected from the group consisting of H, I, L, N, R,and S.

In certain embodiments, the polypeptides comprise the amino acidsequence:

(SEQ ID NO: 146) DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNX ₁PSX ₂QSQGLFGAIAGX ₃ X ₄EGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKX ₅NTQX ₆TAX ₇GKEX ₈NKX ₉ERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDG,wherein X₁ is an amino acid selected from the group consisting of E, I,K, V, A, and T;X₂ is an amino acid selected from the group consisting of I, K, R, T, F,N, S and Y;X₃ is an amino acid selected from the group consisting of D, F, V, Y, A,I, N, S, and T;X₄ is an amino acid selected from the group consisting of I, K, R, T, E,G and V;X₅ is an amino acid selected from the group consisting of M, E, K, V, R,T;X₆ is an amino acid selected from the group consisting of F, I, N, S, T,Y, H, and L;X₇ is an amino acid selected from the group consisting of A, G, I, R, T,V, F, and S;X₈ is an amino acid selected from the group consisting of F, I, N, S, T,Y, G, E, K, M, and V; andX₉ is an amino acid selected from the group consisting of H, I, L, N, R,and S.

In certain embodiments, the polypeptides comprise the amino acidsequence:

(SEQ ID NO: 147) DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNX ₁PSX ₂QSQGLFGAIAGX ₃ X ₄EGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKX ₅NTQX ₆TAX ₇GKEX ₈NKX ₉ERRMKQIEKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGV YQIEG,wherein X₁ is an amino acid selected from the group consisting of E, I,K, V, A, and T;X₂ is an amino acid selected from the group consisting of I, K, R, T, F,N, S and Y;X₃ is an amino acid selected from the group consisting of D, F, V, Y, A,I, N, S, and T;X₄ is an amino acid selected from the group consisting of I, K, R, T, E,G and V:X₅ is an amino acid selected from the group consisting of, M, E, K, V,R, T;X₆ is an amino acid selected from the group consisting of F, I, N, S, T,Y, H, and L;X₇ is an amino acid selected from the group consisting of A, G, I, R, T,V, F, and S;X₈ is an amino acid selected from the group consisting of F, I, N, S, T,Y, G, E, K, M and V; andX₉ is an amino acid selected from the group consisting of H, I, L, N, R,and S.

In certain embodiments, the polypeptides comprise the amino acidsequence:

(SEQ ID NO: 148) DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNX ₁PSX ₂QSQGLFGAIAGX ₃ X ₄EGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKX ₅NTQX ₆TAX ₇GKEX ₈NKX ₉ERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI,wherein X₁ is an amino acid selected from the group consisting of E, I,K, V, A, and T;X₂ is an amino acid selected from the group consisting of I, K, R, T, F,N, S and Y;X₃ is an amino acid selected from the group consisting of D, F, V, Y, A,I, N, S, and T;X₄ is an amino acid selected from the group consisting of I, K, R, T, E,G and V;X₅ is an amino acid selected from the group consisting of M, E, K, V, R,T;X₆ is an amino acid selected from the group consisting of F, I, N, S, T,Y, H, and L;X₇ is an amino acid selected from the group consisting of A, G, I, R, T,V, F, and S;X₈ is an amino acid selected from the group consisting of F, I, N, S, T,Y, G, E, K, M and V: andX₉ is an amino acid selected from the group consisting of H, I, L, N, R,and S.

In certain embodiments, X₁ is K, X₂ is K, X₃ is F, X₄ is T, X₈ is M, X₆is Y, X₇ is I, X₈ is Y, and X₉ is S in SEQ ID NO: 145-148.

The influenza hemagglutinin stem domain polypeptides can be preparedaccording to any technique deemed suitable to one of skill, includingtechniques described below.

Thus, the immunogenic polypeptides of the invention may be synthesizedas DNA sequences by standard methods known in the art and cloned andsubsequently expressed, in vitro or in vivo, using suitable restrictionenzymes and methods known in the art. The present invention thus alsorelates to nucleic acid molecules encoding the above describedpolypeptides. The invention further relates to vectors comprising thenucleic acids encoding the polypeptides of the invention. In certainembodiments, a nucleic acid molecule according to the invention is partof a vector, e.g. a plasmid. Such vectors can easily be manipulated bymethods well known to the person skilled in the art, and can forinstance be designed for being capable of replication in prokaryoticand/or eukaryotic cells. In addition, many vectors can directly or inthe form of an isolated desired fragment there from be used fortransformation of eukaryotic cells and will integrate in whole or inpart into the genome of such cells, resulting in stable host cellscomprising the desired nucleic acid in their genome. The vector used canbe any vector that is suitable for cloning DNA and that can be used fortranscription of a nucleic acid of interest. When host cells are used itis preferred that the vector is an integrating vector. Alternatively,the vector may be an episomally replicating vector.

The person skilled in the art is capable of choosing suitable expressionvectors, and inserting the nucleic acid sequences of the invention in afunctional manner. To obtain expression of nucleic acid sequencesencoding polypeptides, it is well known to those skilled in the art thatsequences capable of driving expression can be functionally linked tothe nucleic acid sequences encoding the polypeptide, resulting inrecombinant nucleic acid molecules encoding a protein or polypeptide inexpressible format. In general, the promoter sequence is placed upstreamof the sequences that should be expressed. Many expression vectors areavailable in the art, e.g. the pcDNA and pEF vector series ofInvitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script fromStratagene, etc, which can be used to obtain suitable promoters and/ortranscription terminator sequences, polyA sequences, and the like. Wherethe sequence encoding the polypeptide of interest is properly insertedwith reference to sequences governing the transcription and translationof the encoded polypeptide, the resulting expression cassette is usefulto produce the polypeptide of interest, referred to as expression.Sequences driving expression may include promoters, enhancers and thelike, and combinations thereof. These should be capable of functioningin the host cell, thereby driving expression of the nucleic acidsequences that are functionally linked to them. The person skilled inthe art is aware that various promoters can be used to obtain expressionof a gene in host cells. Promoters can be constitutive or regulated, andcan be obtained from various sources, including viruses, prokaryotic, oreukaryotic sources, or artificially designed. Expression of nucleicacids of interest may be from the natural promoter or derivative thereofor from an entirely heterologous promoter (Kaufman, 2000). Somewell-known and much used promoters for expression in eukaryotic cellscomprise promoters derived from viruses, such as adenovirus, e.g. theE1A promoter, promoters derived from cytomegalovirus (CMV), such as theCMV immediate early (IE) promoter (referred to herein as the CMVpromoter) (obtainable for instance from pcDNA, Invitrogen), promotersderived from Simian Virus 40 (SV40) (Das et al, 1985), and the like.Suitable promoters can also be derived from eukaryotic cells, such asmethallothionein (MT) promoters, elongation factor 1α (EF-1α) promoter(Gill et al., 2001), ubiquitin C or UB6 promoter (Gill et al., 2001),actin promoter, an immunoglobulin promoter, heat shock promoters, andthe like. Testing for promoter function and strength of a promoter is amatter of routine for a person skilled in the art, and in general mayfor instance encompass cloning a test gene such as lacZ, luciferase,GFP, etc. behind the promoter sequence, and test for expression of thetest gene. Of course, promoters may be altered by deletion, addition,mutation of sequences therein, and tested for functionality, to findnew, attenuated, or improved promoter sequences. According to thepresent invention, strong promoters that give high transcription levelsin the eukaryotic cells of choice are preferred. The constructs may betransfected into eukaryotic cells (e.g. plant, fungal, yeast or animalcells) or suitable prokaryotic expression systems like E. coli usingmethods that are well known to persons skilled in the art. In some casesa suitable ‘tag’ sequence (such as for example, but not limited to, ahis-, myc-, strep-, or flag-tag) or complete protein (such as forexample, but not limited to, maltose binding protein or glutathione Stransferase) may be added to the sequences of the invention to allow forpurification and/or identification of the polypeptides from the cells orsupernatant. Optionally a sequence containing a specific proteolyticsite can be included to afterwards remove the tag by proteolyticdigestion.

Purified polypeptides can be analyzed by spectroscopic methods known inthe art (e.g. circular dichroism spectroscopy, Fourier TransformInfrared spectroscopy and NMR spectroscopy or X-ray crystallography) toinvestigate the presence of desired structures like helices and betasheets. ELISA, Octet and FACS and the like can be used to investigatebinding of the polypeptides of the invention to the broadly neutralizingantibodies described before (CR6261, CR9114, CR8057). Thus, polypeptidesaccording to the invention having the correct conformation can beselected.

The invention further relates to immunogenic compositions comprising atherapeutically effective amount of at least one of the polypeptidesand/or nucleic acids of the invention. The immunogenic compositionspreferably further comprise a pharmaceutically acceptable carrier. Inthe present context, the term “pharmaceutically acceptable” means thatthe carrier, at the dosages and concentrations employed, will not causeunwanted or harmful effects in the subjects to which they areadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (see Remington's Pharmaceutical Sciences, 18thedition, A. R. Gennaro, Ed., Mack Publishing Company [1990];Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]). The term “carrier” refers to a diluent, adjuvant,excipient, or vehicle with which the composition is administered. Salinesolutions and aqueous dextrose and glycerol solutions can e.g. beemployed as liquid carriers, particularly for injectable solutions. Theexact formulation should suit the mode of administration. Thepolypeptides and/or nucleic acid molecules preferably are formulated andadministered as a sterile solution. Sterile solutions are prepared bysterile filtration or by other methods known per se in the art. Thesolutions can then be lyophilized or filled into pharmaceutical dosagecontainers. The pH of the solution generally is in the range of pH 3.0to 9.5, e.g. pH 5.0 to 7.5.

The invention also relates to influenza HA stem domain polypeptides,nucleic acid molecules and/or vectors as described above for use ininducing an immune response against influenza HA protein. The inventionalso relates to methods for inducing an immune response in a subject,the method comprising administering to a subject, a polypeptide, nucleicacid molecule and/or immunogenic composition as described above. Asubject according to the invention preferably is a mammal that iscapable of being infected with an infectious disease-causing agent, inparticular an influenza virus, or otherwise can benefit from theinduction of an immune response, such subject for instance being arodent, e.g. a mouse, a ferret, or a domestic or farm animal, or anon-human-primate, or a human. Preferably, the subject is a humansubject. The invention thus provides methods for inducing an immuneresponse to an influenza virus hemagglutinin (HA), in particular of agroup 1 and/or group 2 influenza A virus, such as an influenza viruscomprising HA of the H1, H2, H3, H4, H5, H7 and/or H10 subtype, and/orof an influenza B virus, in a subject utilizing the polypeptides,nucleic acids and/or immunogenic compositions described herein. In someembodiments, the invention provides methods for inducing an immuneresponse to an influenza virus comprising HA of the H1 subtype, in asubject utilizing the polypeptides, nucleic acids and/or immunogeniccompositions described herein.

In some embodiments, the immune response induced is effective to preventand/or treat an influenza virus infection caused by a group 1 and/orgroup 2 influenza A virus subtypes and/or influenza B viruses. In someembodiments, the immune response induced by the polypeptides, nucleicacids and/or immunogenic compositions described herein is effective toprevent and/or treat an influenza A and/or B virus infection caused bytwo, three, four, five or six subtypes of influenza A and/or B viruses.In some embodiments, the immune response induced is effective to preventand/or treat an influenza virus infection caused by an influenza viruscomprising HA of the H1 subtype.

Since it is well known that small proteins and/or nucleic acid moleculesdo not always efficiently induce a potent immune response it may benecessary to increase the immunogenicity of the polypeptides and/ornucleic acid molecules by adding an adjuvant. In certain embodiments,the immunogenic compositions described herein comprise, or areadministered in combination with, an adjuvant. The adjuvant foradministration in combination with a composition described herein may beadministered before, concomitantly with, or after administration of saidcomposition. Examples of suitable adjuvants include aluminium salts suchas aluminium hydroxide and/or aluminium phosphate; oil-emulsioncompositions (or oil-in-water compositions), including squalene-wateremulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations,such as for example QS21 and Immunostimulating Complexes (ISCOMS) (seee.g. U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762,WO 2005/002620); bacterial or microbial derivatives, examples of whichare monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motifcontaining oligonucleotides, ADP-ribosylating bacterial toxins ormutants thereof, such as E. coli heat labile enterotoxin LT, choleratoxin CT, pertussis toxin PT, or tetanus toxoid TT, Matrix M (Isconova).In addition, known immunopotentiating technologies may be used, such asfusing the polypeptides of the invention to proteins known in the art toenhance immune response (e.g. tetanus toxoid, CRM197, rCTB, bacterialflagellins or others) or including the polypeptides in virosomes, orcombinations thereof. Other non-limiting examples that can be used aree.g. disclosed by Coffman et al. (2010).

In an embodiment, the influenza hemagglutinin stem domain polypeptidesof the invention are incorporated into viral-like particle (VLP)vectors. VLPs generally comprise a viral polypeptide(s) typicallyderived from a structural protein(s) of a virus. Preferably, the VLPsare not capable of replicating. In certain embodiments, the VLPs maylack the complete genome of a virus or comprise a portion of the genomeof a virus. In some embodiments, the VLPs are not capable of infecting acell. In some embodiments, the VLPs express on their surface one or moreof viral (e.g., virus surface glycoprotein) or non-viral (e.g., antibodyor protein) targeting moieties known to one skilled in the art.

In a specific embodiment, the polypeptide of the invention isincorporated into a virosome. A virosome containing a polypeptideaccording to the invention may be produced using techniques known tothose skilled in the art. For example, a virosome may be produced bydisrupting a purified virus, extracting the genome, and reassemblingparticles with the viral proteins (e.g., an influenza hemagglutinin stemdomain polypeptide) and lipids to form lipid particles containing viralproteins.

The invention also relates to the above-described polypeptides, nucleicacids and/or immunogenic compositions for inducing an immune response ina subject against influenza HA, in particular for use as a vaccine. Theinfluenza hemagglutinin stem domain polypeptides, nucleic acids encodingsuch polypeptides, or vectors comprising such nucleic acids orpolypeptides described herein thus may be used to elicit neutralizingantibodies against influenza viruses, for example, against the stemregion of influenza virus hemagglutinin. The invention in particularrelates to polypeptides, nucleic acids, and/or immunogenic compositionsas described above for use as a vaccine in the prevention and/ortreatment of a disease or condition caused by an influenza A virus ofphylogenetic group 1 and/or phylogenetic group 2 and/or an influenza Bvirus. In an embodiment, the vaccine may be used in the preventionand/or treatment of diseases caused by two, three, four, five, six ormore different subtypes of phylogenetic group 1 and/or 2 and/orinfluenza B viruses. In an embodiment, the vaccine may be used in theprevention and/or treatment of influenza infection caused by aninfluenza virus comprising HA of the H1 subtype.

The polypeptides of the invention may be used after synthesis in vitroor in a suitable cellular expression system, including bacterial andeukaryotic cells, or alternatively, may be expressed in vivo in asubject in need thereof, by expressing a nucleic acid coding for theimmunogenic polypeptide. Such nucleic acid vaccines may take any form,including naked DNA, plasmids, or viral vectors including adenoviralvectors.

Administration of the polypeptides, nucleic acid molecules, and/orimmunogenic compositions according to the invention can be performedusing standard routes of administration. Non-limiting examples includeparenteral administration, such as intravenous, intradermal,transdermal, intramuscular, subcutaneous, etc, or mucosaladministration, e.g. intranasal, oral, and the like. The skilled personwill be capable to determine the various possibilities to administer thepolypeptides, nucleic acid molecules, and/or immunogenic compositionsaccording to the invention, in order to induce an immune response. Incertain embodiments, the polypeptide, nucleic acid molecule, and/orimmunogenic composition (or vaccine) is administered more than one time,i.e. in a so-called homologous prime-boost regimen. In certainembodiments where the polypeptide, nucleic acid molecule, and/orimmunogenic composition is administered more than once, theadministration of the second dose can be performed after a time intervalof, for example, one week or more after the administration of the firstdose, two weeks or more after the administration of the first dose,three weeks or more after the administration of the first dose, onemonth or more after the administration of the first dose, six weeks ormore after the administration of the first dose, two months or moreafter the administration of the first dose, 3 months or more after theadministration of the first dose, 4 months or more after theadministration of the first dose, etc, up to several years after theadministration of the first dose of the polypeptide, nucleic acidmolecule, and/or immunogenic composition. It is also possible toadminister the vaccine more than twice, e.g. three times, four times,etc, so that the first priming administration is followed by more thanone boosting administration. In other embodiments, the polypeptide,nucleic acid molecule, and/or immunogenic composition according to theinvention is administered only once.

The polypeptides, nucleic acid molecules, and/or immunogeniccompositions may also be administered, either as prime, or as boost, ina heterologous prime-boost regimen.

The invention further provides methods for preventing and/or treating aninfluenza virus disease in a subject utilizing the polypeptides, nucleicacids and/or compositions described herein. In a specific embodiment, amethod for preventing and/or treating an influenza virus disease in asubject comprises administering to a subject in need thereof aneffective amount of a polypeptide, nucleic acid and/or immunogeniccomposition, as described above. A therapeutically effective amountrefers to an amount of the polypeptide, nucleic acid, and/or compositionas defined herein, that is effective for preventing, ameliorating and/ortreating a disease or condition resulting from infection by a group 1 or2 influenza A virus, and/or an influenza B virus, preferably a diseaseresulting from infection by an influenza A virus comprising HA of the H1subtype. Prevention encompasses inhibiting or reducing the spread ofinfluenza virus or inhibiting or reducing the onset, development orprogression of one or more of the symptoms associated with infection byan influenza virus. Ameloriation as used in herein may refer to thereduction of visible or perceptible disease symptoms, viremia, or anyother measurable manifestation of influenza infection.

Those in need of treatment include those already inflicted with acondition resulting from infection with a group 1 or a group 2 influenzaA virus, or an influenza B virus, as well as those in which infectionwith influenza virus is to be prevented. The polypeptides, nucleic acidsand/or compositions of the invention thus may be administered to a naivesubject, i.e., a subject that does not have a disease caused byinfluenza virus infection or has not been and is not currently infectedwith an influenza virus infection, or to subjects that already areand/or have been infected with an influenza virus.

In an embodiment, prevention and/or treatment may be targeted at patientgroups that are susceptible to influenza virus infection. Such patientgroups include, but are not limited to e.g., the elderly (e.g. ≥50 yearsold, ≥60 years old, and preferably ≥65 years old), the young (e.g. ≤5years old, ≤1 year old), hospitalized patients and patients who havebeen treated with an antiviral compound but have shown an inadequateantiviral response.

In another embodiment, the polypeptides, nucleic acids and/orimmunogenic compositions may be administered to a subject in combinationwith one or more other active agents, such as existing, or futureinfluenza vaccines, monoclonal antibodies and/or antiviral agents,and/or antibacterial, and/or immunomodulatory agents. The one or moreother active agents may be beneficial in the treatment and/or preventionof an influenza virus disease or may ameliorate a symptom or conditionassociated with an influenza virus disease. In some embodiments, the oneor more other active agents are pain relievers, anti-fever medications,or therapies that alleviate or assist with breathing. Dosage regimens ofthe polypeptides and/or nucleic acid molecules of the invention can beadjusted to provide the optimum desired response (e.g., a therapeuticresponse). A suitable dosage range may for instance be 0.1-100 mg/kgbody weight, preferably 1-50 mg/kg body weight, preferably 0.5-15 mg/kgbody weight. The precise dosage of the polypeptides and/or nucleic acidmolecules to be employed will e.g. depend on the route ofadministration, and the seriousness of the infection or disease causedby it, and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedoses vary depending target site, physiological state of the patient(including age, body weight, health), and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages are optimally titrated to optimize safety andefficacy.

The polypeptides of the invention may also be used to verify binding ofmonoclonal antibodies identified as potential therapeutic candidates. Inaddition, the polypeptides of the invention may be used as diagnostictool, for example to test the immune status of an individual byestablishing whether there are antibodies in the serum of suchindividual capable of binding to the polypeptide of the invention. Theinvention thus also relates to an in vitro diagnostic method fordetecting the presence of an influenza infection in a patient saidmethod comprising the steps of a) contacting a biological sampleobtained from said patient with a polypeptide according to theinvention; and b) detecting the presence of antibody-antigen complexes.

The polypeptides of the invention may also be used to identify newbinding molecules or improve existing binding molecules, such asmonoclonal antibodies and antiviral agents.

The invention is further illustrated in the following examples andfigures. The examples are not intended to limit the scope of theinvention in any way.

EXAMPLES Example 1: Stem Based Polypeptides as Described inPCT/EP2014060997

PCT/EP2012/073706 discloses influenza hemagglutinin stem domainpolypeptides, compositions and vaccines and methods of their use in thefield of prevention and/or treatment of influenza. PCT/EP2014/060997discloses additional sequences of stem domain polypeptides derived fromthe full length HA of H1N1 A/Brisbane/59/2007 (SEQ ID NO: 1), which wereobtained by site-directed mutation of H1-mini2-cluster1+5+6-GCN4 (SEQ IDNO: 2) and which also stably presented the broadly neutralizing epitopeof CR6261 (Throsby et al, 2009; Ekiert et al 2010) and/or CR9114.

H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2) was derived from the fulllength HA of H1N1 A/Brisbane/59/2007 (SEQ ID NO: 1) by taking thefollowing steps:

-   -   1. Removal of the cleavage site in HA0. Cleavage of wild type HA        at this site results in HA1 and HA2. The removal can be achieved        by mutation of R to Q at the P1 position (see e.g. Sun et al,        2010 for an explanation of the nomenclature of the cleavage site        (position 343 in SEQ ID NO: 1).    -   2. Removal of the head domain by deleting amino acids 53 to 320        from SEQ ID NO; 1. The remaining N- and C-terminal parts of the        sequence were joined by a four residue flexible linker, GGGG.    -   3. Increasing the solubility of the loop (between the A-helix        and the CD helix) formed by (the equivalent of) residues 402 to        418 in H1 A/Brisbane/59/2007 (SEQ ID NO: 1) in order to both        increase the stability of the pre-fusion conformation and to        destabilize the post-fusion conformation of the modified HA. In        H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2) mutations F406S,        V409T, F413G and L416S (numbering refers to SEQ ID NO: 1) were        introduced    -   4. Introducing a disulfide bridge between amino acids at        position 324 and 436 in H1 A/Brisbane/59/2007; this is achieved        by introducing mutations R324C and Y436C. (numbering refers to        SEQ ID NO: 1)    -   5. Introducing the GCN4 derived sequence MKQIEDKIEEIESKQ (SEQ ID        NO: 5), that is known to trimerize, at position 419-433        (numbering refers to SEQ ID NO: 1).

In certain embodiments, the sequence of the transmembrane andintracellular domain was deleted from position (or the equivalentthereof, as determined from sequence alignment) 514, 515, 516, 517, 518,519, 520, 521, 522, 523, 524, 525, 526, 526, 527, 528, 529, or 530 ofHA2 to the C-terminus of HA2 (numbering according to SEQ ID NO: 1) sothat a secreted (soluble) polypeptide was produced following expressionin cells. The soluble polypeptide was further stabilized by introducinga sequence known to form trimeric structures, i.e. the foldon sequenceGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 3), optionally connected througha short linker, as described above. The linker may optionally contain acleavage site for processing afterwards according to protocols wellknown to those skilled in the art. To facilitate purification anddetection of the soluble form a tag sequence may be optionally added,e.g. a histidine tag (HHHHHH (SEQ ID NO: 15) or HHHHHHH (SEQ ID NO: 16)or a FLAG tag (DYKDDDDK; SEQ ID NO: 22) or combination of these,optionally connected via short linkers. The linker may optionallycontain (part of) a proteolytic cleavage site, e.g. LVPRGS (SEQ ID NO:23) (thrombin) or IEGR (SEQ ID NO: 24) (Factor X) for processingafterwards according to protocols well known to those skilled in theart. The processed proteins are also encompassed in the invention.

An example of such a C-terminal sequence combining FLAG-tag, thrombincleavage site, foldon, and His sequences is SEQ ID NO: 4FLAG-thrombin-foldon-His. This sequence was combined with a soluble formof H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2) sequence to create theparental sequence (SEQ ID NO: 6) that was used to create novelpolypeptides of the invention by mutagenesis. This sequence does notcontain the leader sequence corresponding to amino acids 1-17 of SEQ IDNO: 1 and 2.

The stem domain polypeptides thus were created by deleting the part ofthe hemagglutinin sequence that encodes the head domain of the moleculeand reconnecting the N- and C-terminal parts of the sequence on eitherside of the deletion through a linker as described in PCT/2012/073706and above. The removal of the head domain leaves part of the moleculethat was previously shielded from the aqueous solvent exposed,potentially destabilizing the structure of the polypeptides of theinvention. For this reason residues in the B-loop (in particular aminoacid residue 406 (F and S in SEQ ID NO: 1 and 2, respectively), 409 (Vand T) 413 (F and G) and 416 (L and S) were mutated in variouscombinations using parental sequence SEQ ID NO: 6 as the starting point.SEQ ID NO: 6 was created from H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2)by removing the leader sequence, and replacing residues 520-565 with aFlag-thrombin-foldon—his sequence (SEQ ID NO: 4).

Similarly, in the area around the fusion peptide a number of hydrophobicresidues are exposed to the solvent, caused by the fact that, unlike thenative full length HA, the polypeptides cannot be cleaved and undergothe associated conformational change that buries the hydrophobic fusionpeptide in the interior of the protein. To address this issue some orall of the residues 1337, I1340, F352 and 1353 in SEQ ID NO: 2 were alsomutated.

This way, the soluble forms of HA stem polypeptides 74H9 (SEQ ID NO:57), 127H1 (SEQ ID NO: 55), 71H2 (SEQ ID NO: 61), 86B4 (SEQ ID NO: 56),115A1 (SEQ ID NO: 60), 2201C9 (SEQ ID NO: 63), 55G7 (SEQ ID NO: 59),113E7 (SEQ ID NO: 64), 6E12 (SEQ ID NO: 58), 181H9 (SEQ ID NO: 62) werecreated.

DNA sequences encoding the polypeptides described above were transformedinto Pichia pastoris or transfected into HEK293F cells using protocolswell known to persons skilled in the art. Constructs used for expressionin mammalian cells contained the HA leader sequence (residue 1-17 in SEQID NO: 1 and 2), whereas in constructs used for expression in P.pastoris the HA leader sequence was replaced with the yeast alpha factorleader sequence (SEQ ID NO: 7). In this way expressed protein aredirected towards the cell culture medium thus allowing binding andexpression to be determined without further purification of thepolypeptides of the invention. All sequences contained theFLAG-foldon-HIS C-terminal sequence (SEQ ID NO: 4).

Monoclonal antibody binding (CR6261, CR9114, CR8020) to the polypeptideswas determined by ELISA. To this end ELISA plates were treated overnightwith a 2 μg/ml monoclonal antibody solution (20 μl/well) at 4° C. Afterremoval of the antibody solution the remaining surface was blocked with4% solution of non-fat dry milk powder in PBS for a minimum of 1 h atroom temperature. After washing of the plates, 20 μl of cell culturemedium (neat or diluted) was added to each well and incubated for atleast 1 h at room temperature. ELISA plates were then washed and 20 μlof anti-FLAG-HRP antibody solution (Sigma A8952, 2000 times diluted in4% non-fat dry milk in PBS-Tween) was added. After incubation (1 h atroom temperature) plates were washed once more, and 20 μl luminescentsubstrate (Thermoscientific C#34078) was added to develop the signal.Alternatively, a colorimetric detection method can be used to developthe signal.

Expression of polypeptides of the invention was determined from ahomogeneous time-resolved fluorescence assay (for a general descriptionsee e.g. Degorce et al., Curr. Chem. Genomics 2009 3: 22-32). To thisend a mixture of Terbium (Tb) labeled anti-FLAG monoclonal antibody(donor) and Alexa488 labeled anti-His monoclonal antibody (acceptor)(HTRF solution) was prepared by adding 210.5 μl Anti-FLAG-TB (stocksolution 26 μg/ml) and 1.68 ml of anti-HIS-488 (stock solution 50 μg/ml)to 80 ml of a 1 to 1 mixture of culture medium and 50 mM HEPES+0.1% BSA.19 μl of HTRF solution was added to each well of an ELISA plate and 1 μlof culture medium was added. Upon excitation and after a delay to allowinterfering short-lived background signals arising from other compounds(proteins, media components etc) to decay, the ratio of fluorescenceemission at 520 and 665 nm was determined. This is a measure of totalprotein content in the sample and is used to normalize the mAb bindingsignals between different experiments.

The polypeptides listed in Table 3 and 4 were expressed in P. Pastorisfollowing protocols well known to those skilled in the art. Culturemedium was collected and binding to CR6261 binding of and expression ofthe stem domain polypeptides was determined as described above. Sincethe response in the binding assay scales with the concentration ofexpresses protein, ELISA binding signal was normalized for proteinexpression by comparing the ratio of binding signal over the signal inthe HTRF assay for each expressed sequence. All expressed polypeptidesexhibit higher ratio's of CR6261 binding to HTRF signal compared to theparental sequence of SEQ ID NO: 6.

In addition, the ratio of CR6261 binding to HTRF signals was calculatedand compared to the ratio calculated for the parental sequence SEQ IDNO: 6. The results are listed in column 5 of table 3 and 4; allexpressed proteins exhibit higher ratios, indicating that the stempolypeptides described above show increased binding of CR6261.

Example 2: Design and Characterization of Polypeptides of the Invention

The polypeptides of the present invention contain sequenceRMKQIEDKIEEIESK (SEQ ID NO: 20) or RMKQIEDKIEEIESKQK (SEQ ID NO: 21)derived from yeast transcriptional activator protein GCN4, in the CDhelix. This sequence has a high propensity to form helical secondarystructures and can enhance in this way overall stability of thepolypeptide of the invention. According to the present invention, it hassurprisingly been found that stability and aggregation state of thepolypeptides of the invention is dependent on the exact location andsequence of the GCN4 derived sequence in the primary sequence of thepolypeptides of the invention.

Thus, here we describe a novel set of polypeptides of the inventionwhere sequence RMKQIEDKIEEIESK (SEQ ID NO: 20) is introduced at position419-433 (numbering according to SEQ ID NO: 1; for example SEQ ID NO. 81to 110) or sequence RMKQIEDKIEEIESKQK (SEQ ID NO: 21) is introduced atposition 417-433 (for example SEQ ID NO 111 to 140).

To this end, the polypeptides described in Example 1, i.e 74H9 (SEQ IDNO: 57), 127H1 (SEQ ID NO: 55), 71H2 (SEQ ID NO: 61), 86B4 (SEQ ID NO:56), 115A1 (SEQ ID NO: 60), 2201C9 (SEQ ID NO: 63), 55G7 (SEQ ID NO:59), 113E7 (SEQ ID NO: 64), 6E12 (SEQ ID NO: 58), 181H9 (SEQ ID NO: 62)were modified, using techniques of molecular biology well known to thoseskilled in the art, to create sequences 74H9-t2 (SEQ ID NO: 83), 127H-t2(SEQ ID NO: 81), 71H2-t2 (SEQ ID NO: 87), 86B4-t2 (SEQ ID NO: 82),115A1-t2 (SEQ ID NO: 86), 220C9-t2 (SEQ ID NO: 89), 55G7-t2 (SEQ ID NO:85), 113E7-t2 (SEQ ID NO: 90), 6E12-t2 (SEQ ID NO: 84), 181H9-t2 (SEQ IDNO: 88) containing sequence RMKQIEDKIEEIESK (SEQ ID NO: 20) at position419-433.

In a similar manner sequences 74H9-t3 (SEQ ID NO: 113), 127H1-t3 (SEQ IDNO: 111), 71H2-t3 (SEQ ID NO: 117), 86B4-t3 (SEQ ID NO: 112), 115A1-t3(SEQ ID NO: 116), 2201C9-t3 (SEQ ID NO: 119), 55G7-t3 (SEQ ID NO: 115),113E7-t3 (SEQ ID NO: 120), 6E12-t3 (SEQ ID NO: 114), 181H9-t3 (SEQ IDNO: 118) containing sequence RMKQIEDKIEEIESKQK (SEQ ID NO: 21) atposition 417-433 were created.

Polypeptides of the invention can be created on the basis of thesequence of HA molecules from different viral strains. SEQ ID NO:149-155 for example describe polypeptides of the invention based on theHA sequence of the H1N1 A/California/07/09 strain.

As described before, soluble polypeptides of the invention can becreated by removing the C-terminal part of the HA based sequences forexample from residue 519, 520, 521, 522, 523, 524, 525, 526, 527, 526,528, 529, or 530 of the HA2 domain to the C-terminus of the HA2 domain(numbering according to SEQ ID NO: 1).

The polypeptide scan further be stabilized by introducing a sequenceknown to form trimeric structures, i.e GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQID NO: 3), optionally connected through a linker. The linker mayoptionally contain a cleavage site for processing afterwards accordingto protocols well known to those skilled in the art. To facilitatepurification of the soluble form a tag sequence may be added, e.g. a histag (HHHHHHH (SEQ ID NO: 16) or HHHHHH (SEQ ID NO: 15)) or FLAG tag(DYKDDDDK) (SEQ ID NO: 22) or a combination of these, optionallyconnected via short linkers. The linker may optionally contain (part of)a proteolytic cleavage site, e.g. IEGR (SEQ ID NO: 24) (Factor X) orLVPRGS (SEQ ID NO: 23) (thrombin) for processing afterwards according toprotocols well known to those skilled in the art. The processed proteinsare also encompassed in the invention.

Soluble forms of the polypeptides of SEQ ID NO 55-64 and 81-90 werecreated by replacement of the equivalent of residue 519-565 (numberingrefers to SEQ ID NO: 1) with sequence RSLVPRGSPGHHHHHH, containing botha modified thrombin cleavage site and a 6 histidine tag (SEQ ID NO: 15)and were expressed in HEK293F cells following protocols well known tothose skilled in the art.

For reasons of comparison, soluble forms of H1-mini2-cluster1+5+6-GCN4t2(SEQ ID NO:52) and H1-mini2-cluster1+5+6-GCN4t3 (SEQ ID NO: 53). Culturemedium was collected and binding to CR6261, CR9114 was detected by asandwich ELISA, using coated mAb CR6261 or CR9114 to capture thepolypeptide of the invention directly from the culture medium and aHorse Radish Peroxidase (HRP) conjugated antibody directed against theC-terminal his-tag for detection purposes. Alternatively, biotinylatedCR9114 in combination with HRP-conjugated streptavidin was used fordetection of CR9114 captured polypeptides of the invention in a sandwichELISA. This format allows the detection of the presence of multimericforms of polypeptides of the invention. All polypeptides of theinvention tested were capable of binding to CR9114 (FIGS. 2 A and B,FIGS. 3 A and B and FIGS. 4A and B) and CR6261 (FIGS. 2 C and D, FIGS. 3C and D, FIGS. 4 C and D) as determined by ELISA. Increased levels ofmultimerization as detected by the CR9114 capture—biotinylated CR9114detection sandwich ELISA were observed for s55G7-t2 (SEQ ID NO: 95),s86B4-t2 (SEQ ID NO: 92), s115A1-t2 (SEQ ID NO: 96), s127H1-t2 (SEQ IDNO: 91), s113E7-t2 (SEQ ID NO: 100), s220C9-t2 (SEQ ID NO: 99), s71H2-t3(SEQ ID NO: 127), s127H1-t3 (SEQ ID NO: 121), s74H9-t3 (SEQ ID NO: 123)as shown in FIGS. 2 E and F, FIGS. 3 E and F and FIGS. 4 E and F.

In order to obtain a highly pure preparations of polypeptides of theinvention for further characterization, HEK293F cells were transfectedwith expression vector pcDNA2004 containing the genes encoding solubleforms of 127H1-t2 (SEQ ID NO: 81), 86B4-t2 (SEQ ID NO: 82) and 55G7-t2(SEQ ID NO: 85). It will be understood by the skilled person that theleader sequence (or signal sequence) that directs transport of a proteinduring production (corresponding to amino acids 1-17 of SEQ ID NO: 1)will not be present in the secreted final polypeptide.

To produce the polypeptides of the invention 1.0*10⁶ vc/mL were seededby spinning down HEK293F cells (Invitrogen) at 300 g for 5 min andresuspending in 300 mL pre-warmed Freestyle™ medium per SF1000 flask.This culture was incubated for 1 hour at 37° C., 10% CO2 at 110 rpm in amultitron incubator. After 1 hour the plasmid DNA was pipetted in 9.9 mLOptimem medium to a concentration of 1.0 μg/mL in the 300 mL culturevolume. In parallel 440 μL 293Fectin® was pipetted in 9.9 mL Optimemmedium and incubated for 5 minutes at room temperature. After 5 minutesthe plasmid DNA/Optimem mix was added to the 293Fectin®/Optimem mix andincubated at room temperature for 20 minutes. After the incubation theplasmid DNA/293Fectin® mix was added drop wise to the cell suspension.The transfected cultured was incubated at 37° C., 10% CO2 and 110 rpm ina multitron incubator. At day 7 cells were separated from the culturemedium by centrifugation (30 minutes at 3000 g), while the supernatantcontaining the soluble polypeptides of the invention was filtrated overa 0.2 μm bottle top filter for further processing. For purificationpurposes 1500 ml (s127H1_t2), 1800 ml (s86B4_t2), and 2400 ml (s55G7_t2)of culture supernatant was applied to a 24 ml N1 Sepharose HP column,pre-equilibrated in wash buffer (20 mM TRIS, 500 mM NaCl, pH 7.8).Following a washing step with 10 mM Imidazole in wash buffer the boundpolypeptides of the invention were eluted with a step-wise gradient of300 mM imidazole in wash buffer. The elution peaks were collected,concentrated, and applied to a size exclusion column for furtherpurification (Superdex 200). Elution profiles are shown in FIG. 5. For55G7-t2 and 127H1-t2 fractions were collected, pooled as indicated onthe figure and analyzed by SDS-PAGE (FIG. 6), ELISA and analytical sizeexclusion chromatography combined with multi-angle light scattering toestimate molecular mass (SEC-MALS). ELISA results confirmed binding ofthe polypeptides of the invention to CR6261 and CR9114, but not CR8020.SEC-MALS results are summarized in Table 8.

FIG. 5 and Table 8 indicate that polypeptide of the invention s127H1-t2has a higher yield (˜30 mg protein/1 culture supernatant) compared tos55G7-t2 and s86B4-t2. The majority of the protein exhibits a molecularweight of 62 kDa, which is in between what is expected for a monomer ora dimer. To confirm the aggregation state of the protein the SEC-MALSexperiment was repeated in the presence of Fab-fragments derived fromCR6261, CR9114 and CR8020. Results are shown in FIG. 7 and summarized inTable 8.

The results show that the soluble form of polypeptide of the inventions127H1-t2 forms a complex (as evidenced by the shift of the peak in SECchromatogram) in the presence of the Fab fragments from CR6261 andCR9114, but not with CR8020. This is in line with the specificity of thebinding reactions of the Fab fragments, since CR6261 and CR9114 bind toHA's derived from group 1, whereas CR8020 does not. The size of thecomplex is listed in Table 8, and this indicates that polypeptides127H1-t2 binds one to two Fab fragments, indicating that at least partof the population of purified polypeptide of the invention s127H1-t2 isin dimeric form.

To further analyze the binding reaction between polypeptide of theinvention 127H1-t2 and mAb's CR6261 and CR9114, as well as to confirmthe presence of the conformational epitopes of CR6261 and CR9114 thecomplexation of these antibodies with the purified protein was studiedby biolayer interferometry (Octet Red™, Forte Bio). To this end,biotinylated CR6261, CR9114 and CR8020 were immobilized on streptavidincoated sensors, which subsequently were exposed first to a solution ofthe purified polypeptide of the invention to measure the rate ofassociation and then to a wash solution to measure the rate ofdissociation. The results are shown in FIG. 8.

The immobilized CR6261 and CR9114 both recognize the polypeptide of theinvention as evidenced by the clear responses after exposure to thesoluble form of 127H1-t2 (FIG. 8). To estimate the dissociation constantfor the binding interaction a titration was performed using a 2-folddilution series. Sensors containing immobilized CR6261 or CR9114 wereexposed to soluble s127H1-t2 solutions at concentrations of 40, 20, 10,5, 2.5, 1.3 and 0.63 nM, respectively, and the final response after 6600seconds recorded. The responses were plotted as a function of the stemdomain polypeptide concentration, and a fit to a steady state 1:1binding model was performed, yielding a dissociation constant K_(d) of3.5 nM for the CR6261/stem domain polypeptide complex and 2.3 nM for theCR9114 complex (FIG. 8).

In conclusion polypeptide of the invention s127H1-t2 (SEQ ID NO: 91 isproduced in high quantities and is capable of binding broadlyneutralizing monoclonal antibodies CR6261 and CR9114 with high affinity,confirming the presence of the corresponding neutralizing epitopes inthis stem domain polypeptide. The polypeptide has a propensity to formdimeric structures.

Example 3: Evaluation of Protective Efficacy of a Polypeptide of theInvention in a Lethal Influenza Challenge Model

In order to evaluate the protective efficacy of polypeptides of theinvention s127H1-t2 (SEQ ID NO: 91) in a lethal influenza challengemodel, groups of 10 female BALB/c mice (age 6-8 weeks) were immunized 3times at 3 week intervals with 10 μg of purified s127H1-t2 eitherunadjuvated or adjuvated with 10 μg Matrix-M. As a positive control forthe challenge model, broadly neutralizing antibody monoclonal antibodyCR6261 (15 mg/kg) was administered i.m. 1 day prior to challenge, whileimmunization with PBS served as a negative control. Four weeks after thelast immunization mice were challenged with 25×LD50 heterologouschallenge virus (H1N1 A/Puerto Rico/8/34) and monitored daily (survival,weight, clinical scores) for 3 weeks. Pre-challenge serum is tested inELISA assays for binding to polypeptide of the invention s127H1-t2 thatwas used for immunization (to verify correct immunization), binding tosoluble H1N1 A/Brisbane/59/07 full length HA (to verify recognition offull length HA) and competition with the broadly neutralizing antibodymonoclonal antibody CR9114 for binding to full length HA (to determinewhether induced antibodies bind at close proximity to the broadlyneutralizing CR9114 epitope). The results are shown in FIGS. 9-12.

The results show that the experiment is valid since all mice in the PBScontrol group succumb to infection at day 7 post challenge, whereas thepositive control group (15 mg/kg CR6261, 1 day before challenge) isfully protected (FIG. 9). In contrast to the PBS treated mice, 3 out of10 of the mice immunized with the unadjuavted polypeptide of theinvention s127H1-t2 (SEQ ID NO: 91) and 10 out of 10 of the miceimmunized with the adjuvated polypeptide of the invention survive thelethal challenge (See FIG. 10). Compared to the PBS control group,increased survival proportion, increased survival time and reducedclinical score are observed for the groups immunized with polypeptide ofthe invention s127H1-t2. The differences are most pronounced for thegroup receiving the adjuvated polypeptide of the invention, but are alsoobserved for the group receiving the unadjuvated polypeptide.

The ELISA data using s127H1-t2 or the soluble full length HA as theantigen indicate that the polypeptide of the invention s127H1 isimmunogenic and induces antibodies that are capable of recognizing fulllength HA regardless of the use of an adjuvant (FIGS. 11 A and B).

To further understand the immunological response to the immunization acompetition binding ELISA was performed. To this end plate bound fulllength HA is incubated with serial diluted serum samples, after whichCR9114-biotin at a predetermined titrated concentration is added. Afterfurther incubation, the amount of CR9114-biotin bound is quantifiedusing streptavin-conjugated horse radish peroxidase following protocolswell known in the art. Data are analysed using linear regression of ODversus log dilution, expressed as ‘slope OD’ (ΔOD/10 fold dilution). Thedata show that detectable levels of antibodies that are capable ofcompeting for binding with the broadly neutralizing antibody CR9114 areinduced by immunization with adjuvated polypeptides of the invention, asindicated by the elevated levels of competition observed in FIG. 12A. Asa comparison levels induced by unlabeled CR9114 (i.e. self-competition)and the non-binding monoclonal antibodies CR8020 and CR-JB, bothserially diluted from 5 μg/ml starting concentration are indicated in aseparate graph. In conclusion we have shown that immunization withpolypeptides of the invention s127H1-t2 (SEQ ID NO: 91) can protect miceagainst lethal infection with influenza. The polypeptide is immunogenicand induces antibodies that can bind to full length HA. When thepolypeptide of the invention is used in combination with an adjuvant, atleast part of the induced detectable antibodies bind at, or close to,the epitope of the broadly neutralizing epitope of monoclonal antibodyCR9114.

Example 4: Evaluation of Protective Efficacy of a Polypeptide of theInvention in a Lethal Influenza Challenge Model

In order to further evaluate the protective efficacy of polypeptides ofthe invention s127H1-t2 (SEQ ID NO: 91) in a lethal influenza challengemodel, groups of 10 female BALB/c mice (age 6-8 weeks) were immunized 1,2 and 3 times at 3 week intervals with 30 μg of purified s127H1-t2adjuvated with 10 μg Matrix-M. As a positive control for the challengemodel, broadly neutralizing antibody monoclonal antibody CR6261 (15mg/kg) was administered i.v. 1 day prior to challenge, whileimmunization with PBS served as a negative control. Four weeks after thelast immunization mice were challenged with 25×LD50 heterologouschallenge virus (H1N1 A/Puerto Rico/8/34) and monitored daily (survival,weight, clinical scores) for 3 weeks. Pre-challenge serum obtained 4weeks after the final immunization was tested in ELISA assays forbinding to polypeptide of the invention s127H1-t2 that was used forimmunization (to verify correct immunization), binding to soluble H1N1A/Brisbane/59/07 full length HA (to verify recognition of full lengthHA) and competition with the broadly neutralizing antibody monoclonalantibody CR9114 for binding to full length HA (to determine whetherinduced antibodies bind at close proximity to the broadly neutralizingCR9114 epitope). The results are shown in FIGS. 13-18.

The results show that the experiment is valid since all mice in the PBScontrol group succumbed to infection at day 7 post challenge, whereasthe positive control group (15 mg/kg CR6261, 1 day before challenge) wasfully protected (FIG. 13A). Mice immunized once with s127H1-t2 (SEQ IDNO: 91) all succumbed to infection between day 7 and 9 (FIG. 14A). Incontrast, after two immunizations 8 out of 10 mice survived, and after 3immunizations all mice (10 out of 10) survived the lethal challenge(FIG. 14B,C). Also body weight loss was reduced for groups immunizedmultiple times with lowest percentages observed for animals immunizedthree times (FIG. 15B,C). Compared to the PBS control group,statistically significant increased survival proportion, increasedsurvival time, reduced body weight loss and reduced clinical score (seeFIG. 16B,C) were observed for the groups immunized two or three timeswith polypeptide of the invention s127H1-t2

The ELISA data from pre-challenge timepoints 4 week after the finalimmunization using s127H1-t2 (FIG. 17A) or the soluble full length HA(FIG. 17B) as the antigen indicate that the polypeptide of the inventions127H1 is immunogenic and induces antibodies that are capable ofrecognizing full length HA even after one immunization, although levelsare significantly higher after two and three immunizations. Using theCR9114 competition binding assay described above detectable levels ofantibodies that are capable of competing for binding with the broadlyneutralizing antibody CR9114 were induced after two and threeimmunizations with polypeptide of the invention s127H1-t2 (SEQ ID NO:91) (FIG. 18A). As a comparison levels induced by unlabeled CR9114 (i.e.self-competition) and the non-binding monoclonal antibodies CR8020 andCR-JB, both serially diluted from 5 μg/ml starting concentration areindicated in a separate graph (FIG. 18B).

In conclusion we have shown that two and three times immunization withpolypeptide of the invention s127H1-t2 (SEQ ID NO: 91) can protect miceagainst lethal infection with influenza. The polypeptide is immunogenicand induces antibodies that can bind to full length HA. At least part ofthe induced antibodies bind at, or close to, the epitope of the broadlyneutralizing epitope of monoclonal antibody CR9114.

Example 5: Evaluation of Protective Efficacy of a Polypeptide of theInvention in a Lethal Heterosubtypic HSN1 Influenza Challenge Model

In order to further evaluate the protective efficacy of polypeptides ofthe invention s127H1-t2- (SEQ ID NO: 91) in a lethal H5N1 influenzachallenge model, groups of 8-12 female BALB/c mice (age 6-8 weeks) wereimmunized 3 times at 3 week intervals with 30 μg of purified s127H1-t2adjuvated with 10 μg Matrix-M. As a positive control for the challengemodel, broadly neutralizing antibody monoclonal antibody CR6261 (15mg/kg) was administered i.v. I day prior to challenge, whileimmunization with PBS served as a negative control. Four weeks after thelast immunization mice were challenged with 12.5×LD50 heterosubtypicchallenge virus (H5N1 A/Hong Kong/156/97) and monitored daily (survival,weight, clinical scores) for 3 weeks.

The results show that the experiment is valid since all mice in the PBScontrol group succumb to infection between day 8-10 post challenge,whereas the positive control group (15 mg/kg CR6261, 1 day beforechallenge) is fully protected (FIG. 19A). Eight out of 10 (80%) miceimmunized with s127H1-t2 (SEQ ID NO: 91) survive the lethal challenge(FIG. 19B). Mean bodyweight loss is approximately 15% at day 9, butsurviving animals recover and gain bodyweight (FIG. 19C). Medianclinical score is 1.5 at day 3-6, but from day 8 onwards no clinicalsymptoms were observed for surviving mice (FIG. 19D). Compared to thePBS control group, a statistical significant increased survivalproportion, increased survival time, a decrease of body weight loss andreduced clinical scores are observed for the group immunized withpolypeptide of the invention s127H1-t2. In conclusion we have shown thatimmunization with polypeptide of the invention s127H1-t2 (SEQ ID NO: 91)can protect mice against lethal infection with a heterosubtypic H5N1influenza strain.

Example 6: Evaluation of the Breadth of Binding of Sera Elicited ThroughImmunization with a Polypeptide of the Invention

Pre-challenge sera from mice immunized 3 times as described in example 5were also tested for binding against full length HA's from a number ofother group 1 (H1, H5 and H9) and group 2 (H3 and H7) influenza strainsby ELISA following protocols well known in the art (FIG. 20). Theresults demonstrate that antibodies induced with polypeptide of theinvention s127H1-t2 (SEQ ID NO: 91) efficiently recognize epitopespresent in the native sequences of FL HA and that the epitopes to whichthe antibodies bind are conserved among different group 1 influenzastrains including H1, H5 and H9 HA.

Example 7: Evaluation of Protective Efficacy of a Polypeptide of theInvention in a Lethal H1N1 A/Brisbane/59/2007 Influenza Challenge Model

In order to further evaluate the protective efficacy of s127H1-t2 (SEQID NO: 91) in a lethal H1N1 influenza challenge model, groups of 8-18female BALB/c mice (age 6-8 weeks) were immunized 3 times at 3 weekintervals with 30 μg of purified s127H1-t2 adjuvated with 10 μgMatrix-M. As a positive control for the challenge model, broadlyneutralizing antibody monoclonal antibody CR6261 (15 mg/kg) wasadministered i.v. 1 day prior to challenge, while immunization with PBSserved as a negative control. Four weeks after the last immunizationmice were challenged with 12.5×LD50 challenge virus (H1N1A/Brisbane/59/2007) and monitored daily (survival, weight, clinicalscores) for 3 weeks.

The results show that the experiment is valid since all mice in the PBScontrol group succumb to infection between day 7-10 post challenge,whereas the positive control group (15 mg/kg CR6261, 1 day beforechallenge) is fully protected (FIG. 21A). Ten out of 10 mice immunizedwith s127H1-t2 (SEQ ID NO: 91) survive the lethal challenge (FIG. 21B).In addition bodyweight loss is ca 20% on average 5 days post infection(FIG. 21C), but animals recover fully within the 21 days follow-upperiod. Median clinical scores peak at a value of 3 between 2 and 9 dayspost infection but return to baseline level (0) from day 16 postinfection onwards (FIG. 21D). Compared to the PBS control group, astatistical significant increased survival proportion, increasedsurvival time, a decrease of body weight loss and reduced clinicalscores are observed for the group immunized with polypeptide of theinvention s127H1-t2 In conclusion we have shown that immunization withpolypeptide of the invention s127H1-t2 (SEQ ID NO: 91) can protect miceagainst lethal infection with H1N1 A/Brisbane/59/2007.

Example 8: Evaluation of the Presence of Influenza NeutralizingAntibodies in Sera of Mice Immunized with the Polypeptide of theInvention

To further investigate antibody-mediated effector mechanisms that play arole in protection against influenza, pre-challenge sera were tested inpseudoparticles neutralization assay (Alberini et al 2009) using thepseudoparticles derived from H5N1 A/Vietnam/1194/04 as described below.

Pseudoparticle Neutralization Assay

Pseudoparticles expressing FL HA were generated as previously described(Temperton et al., 2007). Neutralizing antibodies were determined usinga single transduction round of HEK293 cells with H5 A/Vietnam/1194/04pseudoparticles encoding luciferase reporter gene, as describedpreviously (Alberini et al 2009), with a few modifications. Briefly,heat-inactivated (30 minutes at 56° C.) pre-challenge serum samples were3-fold serially diluted in growth medium (MEM Eagle with EBSS (Lonza,Basel, Switserland) supplemented with 2 mM L-Glutamine (Lonza), 1%Non-Essential Amino Acid Solution (Lonza), 100 U/ml Pen/Strep (Lonza)and 10% FBS (Euroclone, Pero, Italy)) in triplicate in 96-well flatbottom culture plates and a titrated number of H5 A/Vietnam/1194/04pseudoparticles (yielding 10⁶ Relative Luminescence Units (RLU)post-infection) was added. After 1 h incubation at 37° C., 5% CO₂ 10⁴HEK293 cells were added per well. After 48 h incubation at 37° C., 5%CO₂ luciferase substrate (Britelie Plus, Perkin Elmer, Waltham, Mass.)was added and luminescence measured using a luminometer (Mithras LB 940,Berthold Technologies, Germany) according to manufacturers'instructions.

Pre challenge sera obtained from animals immunized with polypeptide ofthe invention s127H1-t2 (SEQ ID NO: 91) as described in examples 5, 6,and 7 showed detectable neutralization at high serum concentrationsusing the pseudoparticle neutralization assay (FIG. 22). Thisdemonstrates the ability of the polypeptide of the invention to elicitbroadly neutralizing antibodies when used as an immunogen.

Besides direct virus neutralization, Fc-mediated effector mechanisms,such as Antibody Dependent Cellular Cytotoxicity (ADCC) and AntibodyDependent Cellular Phagocytosis (ADCP), contribute substantially toprotection against influenza, with stem-directed bnAbs beingparticularly effective in these mechanisms (DiLillo et al., 2014). Inorder to test whether the antibodies elicited after immunization withpolypeptide of the invention s127H1-t2118long (SEQ ID NO: 186) werecapable of inducing ADCC, we tested pre-challenge sera using an ADCCsurrogate assay (Parekh et al., 2012; Schneuriger et al., 2012; Cheng etal., 2014), adapted for mouse as described below. Antibody DependentCellular Cytotoxicity (ADCC) surrogate assay Human lungcarcinoma-derived A549 epithelial cells (ATCC CCL-185) were maintainedin Dulbecco's modified eagle medium (DMEM) medium supplemented with 10%heat inactivated fetal calf serum at 37° C., 10% CO2. Two days beforethe experiment, A549 cells were transfected with plasmid DNA encoding H5A/Hong Kong/156/97 HA or H1 A/Brisbane/59/2007 HA using Lipofectamine2000 (Invitrogen) in Opti-MEM (Invitrogen). One day before the assay,transfected cells were harvested and seeded in white 96-well plates(Costar) for ADCC, and black clear bottom 96-well plate (BD Falcon) forimaging. After 24 hours, samples were diluted in assay buffer (4%ultra-low IgG FBS (Gibco) in RPMI 1640 (Gibco)) and heat inactivated for30 minutes at 56° C., followed by serial dilution in assay buffer. Forthe ADCC bioassay, A549 cells were replenished with fresh assay bufferand antibody dilutions and ADCC Bioassay Jurkat effector cellsexpressing mouse Fc gamma receptor IV (FcγRIV; Promega) were added tothe cells and incubated for 6 hours at 37° C. at a target-effector ratioof 1:4.5. Cells were equilibrated to room temperature for 15 min beforeBio-Glo Luciferase System substrate (Promega) was added. Luminescencewas read out after 10 minutes on a Synergy Neo (Biotek). Data areexpressed as fold induction of signal in the absence of serum.

Using this assay pre-challenge sera obtained from animals immunized withpolypeptide of the invention s127H1-t2 (SEQ ID NO: 91) as described inexamples 5, 6, and 7 were tested for FcγRIV signaling activity usingtarget cells transfected with FL HA from H5N1 A/Hong Kong/156/97 or H1N1A/Brisbane/59/07 as the source of antigen (FIG. 23). In both cases a 30fold induction is observed at highest serum concentration tested,demonstrating the ability of the polypeptide of the invention to elicitantibodies that activate FcγRIV signaling, indicative for ADCC/ADCPeffector function in mice. These results shown in examples 5-8 show thecapability of polypeptide of the invention s127H1-t2 (SEQ ID NO: 91) isable to elicit stem-targeting, neutralizing and ADCC-mediatingantibodies and protect mice against a lethal challenge with homologous,heterologous and heterosubtypic group 1 influenza strains.

Example 9: Protection from Lethal Challenge with HSN1 A/Hong Kong/156/97by Passive Transfer of Serum from Mice Immunized with Polypeptides ofthe Invention

To determine the contribution of antibodies induced by polypeptides ofthe invention to protection observed, transfer studies were performed.The aim of this study was to assess whether passive transfer (multipledosing) of serum from mice immunized three times with s127H1-t2 (SEQ IDNO: 91) and s127H1-t2long (SEQ ID NO: 101) containing an additionalHis-tag in the presence of an adjuvant (Matrix-M) confers protection toa lethal challenge with H5N1 Influenza A/Hong Kong/156/97.

Groups of female BALB/c donor mice (age 6-8 weeks) were immunized 3times at a 3 week interval with 30 μg s127H1-t2 (SEQ ID NO: 91)s127H1-t2long (SEQ ID NO: 101) containing a C-terminal His-tag adjuvatedwith 10 μg Matrix-M or PBS. Four weeks after the last immunization (d70)serum was isolated, pooled per group and transferred in recipient mice(female BALB/c, age 6-8 weeks, n=10 per group). Each mouse received 400μl serum i.p. on three consecutive days before challenge (d−3, −2 and−1). As a positive control for the challenge model CR6261 (15 mg/kg) wasadministered 1 day prior to challenge (n=8), while injection with PBSserved as a negative control (n=8). On day 0, mice were challenged with12.5×LD50 challenge virus and monitored (survival, weight, clinicalscores) for 3 weeks.

To verify immunogenicity of polypeptides of the invention in donor miceand asses HA-specific antibody levels after transfer of serum intorecipient mice, pooled serum samples of terminal bleeds (d70) of donormice, pooled serum samples of naive recipient mice before serum transfer(d−4) as well as individual serum samples of recipient mice after 3serum transfers just prior to challenge (d0), were tested in ELISA forbinding to FL HA from H1N1 A/Brisbane/59/07.

Results Challenge

-   -   Experiment was valid; all mice in the PBS control group succumb        to infection at or before day 13 post challenge (median 9.5        days), whereas the positive control group (15 mg/kg CR6261, 1        day before challenge) is fully protected (p<0.001).    -   Three serum transfers of serum from Matrix-M adjuvated        polypeptide of the invention SEQ ID NO: 91 immunized mice into        naive recipient mice leads to significant increase in survival        time (p=0.007) and reduction in clinical score (p=0.012),        compared to the PBS serum transfer control group (FIG. 24).    -   Three serum transfers of serum from Matrix-M adjuvated        polypeptide of the invention SEQ ID NO: 101 immunized mice into        naive recipient mice leads to significant increase in survival        proportion (p=0.002), increase in survival time (p<0.001),        decrease in bodyweight loss (p=0.002) and reduction in clinical        score (p<0.001), compared to the PBS serum transfer control        group. (FIG. 24)    -   For the polypeptides of the invention tested FL HA        A/Brisbane/59/07 specific antibody titers after three serum        transfers wee similar to levels obtained after active        immunization (FIG. 25).

CONCLUSION

Serum components (most likely antibodies) induced by 3 timesimmunization with Matrix-M adjuvated polypeptide of the inventions SEQID NO: 91 and 101 can protect mice from lethal challenge with H5N1A/Hong Kong/156/97 (survival percentages are 30 and 78%, respectively).

Example 10: In Vivo Protective Efficacy of Polypeptides of the Inventionin H1N1 A/NL/602/09 Challenge Model in Mice

The protective efficacy of polypeptides of the invention s127H1-t2 (SEQID NO: 91) and s127H1-t2long (SEQ ID NO: 101) containing an additionalHis-tag with Matrix-M in a H1N1 A/NL/602/09 challenge model compared toa PBS control group was determined.

Groups of 10 female BALB/c mice (age 6-8 weeks) were immunized 3 timesat a 3 week interval with 30 μg polypeptides of the invention with 10 μgMatrix-M. As a positive control for the challenge model CR6261 (15mg/kg) was administered 1 day prior to challenge (n=8), while injectionwith PBS served as a negative control (n=18). Four weeks after the lastimmunization mice were challenged with 12.5×LD50 challenge virus andmonitored (survival, weight, clinical scores) for 3 weeks.

To verify immunogenicity of polypeptides of the invention, pre-challengesera (day −1) were tested in ELISA assays for binding to FL HA from H1N1A/Brisbane/59/07. To determine whether induced antibodies bind at closeproximity to the CR9114 epitope, a CR9114 competition ELISA wasperformed. Competition data were expressed as using the slope OD to beable to quantify responses.

Results

-   -   The experiment was valid; all mice in the PBS control group        succumb to infection at or before day 8 post challenge (median 5        days), whereas the positive control group (15 mg/kg CR6261, 1        day before challenge) is fully protected (p<0.001).    -   Three immunizations with Matrix-M adjuvated s127H1-t2 (SEQ ID        NO: 91) and s127H1-t2long (SEQ ID NO: 101) containing an        additional His-tag lead to significant increase in survival        proportion (p<0.001), increase in survival time (p<0.001) and        reduction in clinical score (p<0.001), compared to the PBS        control group (FIG. 26).    -   Three immunizations with Matrix-M adjuvated H1 mini-HA variant        s127H1-t2 (SEQ ID NO: 91) leads to significant decrease in        bodyweight (p<0.001) compared to the PBS control group (FIG.        26).    -   IgG antibody titers to H1N1 A/Brisbane/59/07 FL HA induced by        polypeptides of the invention are significantly higher compared        to PBS for all H1 mini-HA variants tested (p<0.001) (FIG. 27A).    -   H1 mini-HA variant s127H1-t2 (SEQ ID NO: 91) has significantly        higher IgG antibody titers to H1N1 A/Brisbane/59/07 FL HA        compared to s127H1-t2long (SEQ ID NO: 101) containing an        additional His-tag (p=0.021) (FIG. 27A).    -   All Matrix-M adjuvanted polypeptides of the invention tested        have significantly higher CR9114 competition titers compared to        PBS (p<0.001) (FIG. 27B).

CONCLUSION

Matrix-M adjuvated polypeptides of the invention s127H1-t2 (SEQ ID NO:91) and s127H1-t2long (SEQ ID NO: 101) containing an additional His-tagconfer protection against lethal challenge with H1N1 A/NL/602/09, seenas increase in survival proportion, survival duration and reduction ofclinical scores. In addition, Matrix-M adjuvated s127H1-t2 (SEQ ID NO:91) also resulted in a reduced bodyweight loss after lethal challengewith H1N1 A/NL/602/09.

Example 11: Library Screening

PCT/EP2012/073706 discloses influenza hemagglutinin stem domainpolypeptides, compositions and vaccines and methods of their use in thefield of prevention and/or treatment of influenza. Here we describeadditional sequences of stem domain polypeptides derived from the fulllength HA of H1N1 A/Brisbane/59/2007 (SEQ ID NO: 1). The stem domainpolypeptides are obtained by site-directed mutation ofH1-mini2-cluster1+5+6-GCN4t2 (SEQ ID NO: 52) and present the broadlyinfluenza neutralizing epitope of CR6261 (Throsby et al, 2009; Ekiert etal 2010) and/or CR9114.

H1-mini2-cluster1+5+6-GCN4t2 (SEQ ID NO: 52) was derived from the fulllength HA of H1N1 A/Brisbane/59/2007 (SEQ ID NO: 1) by taking thefollowing steps:

-   -   Removal of the cleavage site in HA0. Cleavage of wild type HA at        this site results in HA1 and HA2. The removal can be achieved by        mutation of R to Q at the P1 position (see e.g. Sun et al, 2010        for an explanation of the nomenclature of the cleavage site        (position 343 in SEQ ID NO: 1).    -   Removal of the head domain by deleting amino acids 53 to 320        from SEQ ID NO; 1. The remaining N- and C-terminal parts of the        sequence were joined by a four residue flexible linker, GGGG.    -   Increasing the solubility of the loop (between the A-helix and        the CD helix) formed by (the equivalent of) residues 402 to 418        in H1 A/Brisbane/59/2007 (SEQ ID NO: 1) in order to both        increase the stability of the pre-fusion conformation and to        destabilize the post-fusion conformation of the modified HA. In        H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2) mutations F406S,        V409T, F413G and L416S (numbering refers to SEQ ID NO: 1) were        introduced.    -   Introducing a disulfide bridge between amino acids at position        324 and 436 in H1 A/Brisbane/59/2007; this is achieved by        introducing mutations R324C and Y436C. (numbering refers to SEQ        ID NO: 1).    -   Introducing the GCN4 derived sequence RMKQIEDKIEEIESK (SEQ ID        NO: 20), that is known to trimerize, at position 419-433        (numbering refers to SEQ ID NO: 1).

In certain embodiments, the polypeptides of the invention contain theintracellular sequences of HA and the transmembrane domain. In otherembodiments, the sequence of the transmembrane and intracellular domainhave been deleted from position (or the equivalent thereof, asdetermined from sequence alignment) 519, 520, 521, 522, 523, 524, 525,526, 526, 527, 528, 529, or 530 of HA2 to the C-terminus of HA2(numbering according to SEQ ID NO: 1) so that a secreted (soluble)polypeptide is produced following expression in cells. The solublepolypeptide can be further stabilized by introducing a sequence known toform trimeric structures, i.e. the foldon sequence AYVRKDGEWVLL (SEQ IDNO: 3), optionally connected through a short linker, as described above.The linker may optionally contain a cleavage site for processingafterwards according to protocols well known to those skilled in theart. To facilitate purification and detection of the soluble form a tagsequence may be optionally added, e.g. a histidine tag (HHHHHHH (SEQ IDNO: 16) or HHHHHH (SEQ ID NO: 15) or a FLAG tag (DYKDDDDK; SEQ ID NO:22) or combination of these, optionally connected via short linkers. Thelinker may optionally contain (part of) a proteolytic cleavage site,e.g. LVPRGS (SEQ ID NO: 23) (thrombin) or IEGR (SEQ ID NO: 24) (FactorX) for processing afterwards according to protocols well known to thoseskilled in the art. The processed proteins are also encompassed in theinvention.

An example of such a C-teminal sequence combining FLAG-tag, thrombincleavage site, foldon, and His sequences is SEQ ID NO: 4FLAG-thrombin-foldon-His. This sequence was combined with a soluble formof H1-mini2-cluster1+5+6-GCN4t2 (SEQ ID NO: 51) sequence to create theparental sequence (SEQ ID NO: 156) that was used to create novelpolypeptides of the invention by mutagenesis. This sequence does notcontain the leader sequence corresponding to amino acids 1-17 of SEQ IDNO: 1 and 2.

The stem domain polypeptides are created by deleting the part of thehemagglutinin sequence that encodes the head domain of the molecule andreconnecting the N- and C-terminal parts of the sequence on either sideof the deletion through a linker as described in PCT/2012/073706 andabove. The removal of the head domain leaves part of the molecule thatwas previously shielded from the aqueous solvent exposed, potentiallydestabilizing the structure of the polypeptides of the invention. Forthis reason residues in the B-loop (in particular amino acid residue 406(F and S in SEQ ID NO: 1 and 2, respectively), 409 (V and T) 413 (F andG) and 416 (L and S) were mutated in various combinations using parentalsequence SEQ ID NO: 156 as the starting point. SEQ ID NO: 156 wascreated from H1-mini2-cluster1+5+6-GCN4t2 (SEQ ID NO: 52) by removingthe leader sequence, and replacing residues 520-565 with aFlag-thrombin-foldon—his sequence (SEQ ID NO: 4).

Similarly, in the area around the fusion peptide a number of hydrophobicresidues are exposed to the solvent, caused by the fact that, unlike thenative full length HA, the polypeptides of the invention cannot becleaved and undergo the associated conformational change that buries thehydrophobic fusion peptide in the interior of the protein. To addressthis issue some or all of the residues 1337, I1340, F352 and 1353 in SEQID NO: 156 were also mutated.

Two different sets of mutant polypeptides are disclosed in Table 9. Inall cases these polypeptides contain SEQ ID NO: 20 at position 419-433(numbering refers to SEQ ID NO: 1).

Example 12: Identification, Purification and Characterization of theTrimeric Polypeptides of the Invention

Libraries of polypeptides as described in example 11 (set 1 and set 2)containing SEQ ID NO: 20 at position 419-433 were created. Single clonesinto HEK293F cells and screen culture medium for multimers (CR9114sandwich ELISA), CR6261 binding (ELISA) and protein expression (HTRFassay) were individually transfected. Hits based on CR9114 sandwichassay, CR9114, CR6261, and CR8020 ELISA, and HTRF assay were confirmedand ranked.

Multimerization by crosslinking with primary amine (present in Lysineresidues) specific crosslinker BS3 followed by SDS-PAGE (see below) wasassessed. Because of extensive multimerization, the C-terminalFlag-Foldon-His (FFH) tag sequence was replaced with thrombin cleavagesite and his-tag sequence (TCShis). Subsequently, multimerization ofTCS-his containing sequences (CR9114 sandwich assay, BS3 cross-linking)was re-confirmed, and clones were ranked and selected. Selected cloneswere expressed, purified and characterized.

The cross-linking assay was performed as follows:

-   -   Add cross-linker BS3 (bis(sulfosuccinimidyl)suberate) directly        to culture medium    -   Incubate for 30 min at room temperature.    -   Collect medium and analyze by SDS-PAGE/Western Blot under        reducing (R, 5 mM DTT) and non-reducing (NR) conditions    -   Under reducing conditions only BS3-crosslinked species will        remain covalently linked    -   Detection of mini-HA via Western blotting using a his-tag        specific mAb

Results:

-   -   1. Two libraries of high quality (>90% of ORF correct)        containing SEQ ID NO: 20 at position 419-433 and the expected        sequence variation (>97% randomization) were successfully        created    -   2. A total of 10472 clones (5544 and 4928 from set 1 and 2,        respectively) were evaluated in the primary screen (FIG. 28)    -   3. Clones exhibiting expression >50% of FL HA expression and        binding signals to CR6261 >80% of the signals observed for FL HA        were considered hits; this procedure yielded 703 hits (596 and        107 from library 1 and 2, respectively)    -   4. 658 out of 703 hits were retained after the confirmation        screen    -   5. Crosslinking assay of top 20% hits (111) indicated the        presence of higher order multimers that could potentially        interfere with purification of trimeric species.    -   6. Top 20% confirmed hits (111) were successfully cloned to        replace FFH C-terminus with TCS-his sequence, followed by CR9114        sandwich ELISA and crosslinking assay evaluations    -   7. Cross-linking assays yielded 9 clones that were considered        the most promising trimer candidates (SEQ ID NO: 158 to 166,        Table 11). Based on the CR9114 sandwich ELISA (FIG. 29) three        candidates (2 with TCS-his, 1 with FFH C-terminus) were selected        for expression and purification    -   8. Two of the selected candidates did not express well and        purification was not pursued. Candidate GW1.5E2.FFH (SEQ ID        NO: 158) was purified to homogeneity (7.6 mg total protein;        purity >95%, HP-SEC), following procedures as described in        Example 4.    -   9. Characterization of GW1.5E2.FFH (SEQ ID NO: 158) by SEC-MALS        analysis indicates trimer formation in solution, with 3 Fab        fragments of CR9114 or CR6261 binding per trimer (FIG. 30 and        table below 10). K_(d) ^(app) as determined from bilayer        interferometry measurements (Octet) is 1 nM for both CR6261 and        CR9114. As expected, binding of CR8020 (negative control) could        not be detected by either method.

CONCLUSION

The non-covalent trimeric polypeptide of the invention (GW 1.5E2.FFH,SEQ ID NO: 158) that binds bnAbs CR6261 and CR9114 with high affinity ina 3:1 stoichiometry has been identified.

Example 13: Protective Efficacy of Polypeptide of the Invention sH1Mini-HA GW1.5E2-FFH (SEQ ID NO: 158) in H1N1 A/Brisbane/59/07 MouseModel

The protective efficacy of sH mini-HA GW1.5E2-FFH (SEQ ID NO: 158)adjuvated with Matrix-M in a H1N1 A/Brisbane/59/07 challenge modelcompared to a PBS control group was determined.

Groups of 10 female BALB/c mice (age 6-8 weeks) were immunized 3 timesat a 3 week interval with 30 μg sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158)adjuvated with 10 μg Matrix-M. As a positive control for the challengemodel CR6261 (15 mg/kg) was administered 1 day prior to challenge (n=8),while injection with PBS served as a negative control (n=16). Four weeksafter the last immunization mice were challenged with 12.5×LD50challenge virus and monitored (survival, weight, clinical scores) for 3weeks.

To verify immunogenicity of sH mini-HA GW 1.5E2-FFH (SEQ ID NO: 158),pre-challenge sera (day −1) were tested in ELISA assays for binding toFL HA from H1N1 A/Brisbane/59/07. To determine whether polypeptide ofthe invention sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158) inducedantibodies bind at close proximity to the CR9114 epitope, a CR9114competition ELISA was performed. Competition data were visualized as ‘%competition’, defined as (A−P)/A×100), where A is the maximum OD signalof CR9114 binding to FL HA when no serum is present and P is the ODsignal of CR9114 binding to FL HA in presence of serum at a givendilution or expressed using the slope OD metric to be able to quantifyresponses; for reference CR9114 and CR8020 (starting concentration 5mg/ml) solutions were included.

Results:

-   -   Experiment was valid; all mice in the PBS control group (n=16)        succumb to infection at or before day 10 post challenge (median        8 days), whereas the positive control group (n=8, 15 mg/kg        CR6261, 1 day before challenge) is fully protected (p<0.001).    -   Three immunizations with sH1 mini-HA GW 1.5E2-FFH (SEQ ID        NO: 158) adjuvated with Matrix-M lead to significant increase in        survival proportion (p<0.001), increase in survival time        (p<0.001), decrease in bodyweight loss (p<0.001) and reduction        in clinical score (p<0.001), compared to the PBS control group        (FIG. 31).    -   Pre-challenge IgG antibody titers to H1N1 A/Brisbane/59/07 FL HA        induced by sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158) are        significantly higher compared to PBS (p<0.001) (FIG. 32A).    -   IgG antibody titers to H1N1 A/Brisbane/59/07 FL HA plateau after        two immunizations (not shown).    -   Matrix-M adjuvated polypeptide of the invention sH1 mini-HA        GW1.5E2-FFH (SEQ ID NO: 158) induce significantly higher CR9114        competition titers compared to PBS (p<0.001) (FIG. 32B).

CONCLUSION

Matrix-M adjuvated polypeptide of the invention sH1 mini-HA GW1.5E2-FFH(SEQ ID NO: 158) confers protection against lethal challenge with H1N1A/Brisbane/59/07.

Example 14: Protective Efficacy of Polypeptide of the Invention sH1Mini-HA GW1.5E2-FFH (SEQ ID NO: 158) in a H5N1 A/Hong Kong/156/97 MouseModel

The protective efficacy of leading H1 mini-HA variants adjuvated withMatrix-M in a H5N1 A/Hong Kong/156/97 challenge model compared to a PBScontrol group was determined.

Groups of 10 female BALB/c mice (age 6-8 weeks) were immunized 3 timesat a 3 week interval with 30 μg polypeptide of the invention sH1 mini-HAGW1.5E2-FFH (SEQ ID NO: 158) adjuvated with 10 μg Matrix-M. As apositive control for the challenge model CR6261 (15 mg/kg) wasadministered 1 day prior to challenge (n=8), while injection with PBSserved as a negative control (n=16). Four weeks after the lastimmunization mice were challenged with 12.5×LD50 challenge virus andmonitored (survival, weight, clinical scores) for 3 weeks.

To verify immunogenicity of polypeptide of the invention sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158), pre-challenge sera (day −1) were tested inELISA assays for binding to FL HA from H1N1 A/Brisbane/59/07. Todetermine whether mini-HA induced antibodies bind at close proximity tothe CR9114 epitope, a CR9114 competition ELISA was performed.Competition data were visualized as ‘% competition’, defined as(A−P)/A×100), where A is the maximum OD signal of CR9114 binding to FLHA when no serum is present and P is the OD signal of CR9114 binding toFL HA in presence of serum at a given dilution or expressed using theslope OD metric to be able to quantify responses, for reference CR9114and CR8020 (starting concentration 5 μg/ml) solutions were included.

Results:

-   -   Experiment was valid; 15 out of 16 mice in the PBS control group        succumb to infection at or before day 9 post challenge (median 9        days), whereas the positive control group (n=8, 15 mg/kg CR6261,        1 day before challenge) is fully protected (p<0.001).    -   Three immunizations polypeptide of the invention sH1 mini-HA        GW1.5E2-FFH (SEQ ID NO: 158) adjuvated with Matrix-M lead to        significant increase in survival proportion (p<0.001), increase        in survival time (p<0.001), decrease in bodyweight loss        (p<0.001) and reduction in clinical score (p<0.001), compared to        the PBS control group (FIG. 33).    -   Pre-challenge IgG antibody titers to H1N1 A/Brisbane/59/07 FL HA        induced by polypeptide of the invention sH1 mini-HA GW1.5E2-FFH        (SEQ ID NO: 158) are significantly higher compared to PBS        (p<0.001) (FIG. 34A).    -   Matrix-M adjuvated polypeptide of the invention sH mini-HA        GW1.5E2-FFH (SEQ ID NO: 158) induce significantly higher CR9114        competition titers compared to PBS (p<0.001) (FIG. 34B).

CONCLUSION

Matrix-M adjuvated polypeptide of the invention sH1 mini-HA GW1.5E2-FFH(SEQ ID NO: 158) confers heterosubtypic protection against lethalchallenge with H5N1 A/Hong Kong/156/97.

Example 15: Protective Efficacy of Polypeptide of the Invention sH1Mini-HA GW1.5E2-FFH (SEQ ID NO: 158) in a H1N1 A/Puerto Rico/8/34 MouseModel

The protective efficacy of polypeptide of the invention sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158) adjuvated with Matrix-M in a H1N1 A/PuertoRico/8/1934 challenge model compared to a PBS control group wasdetermined.

Groups of 10 female BALB/c mice (age 6-8 weeks) were immunized 3 timesat a 3 week interval with 30 μg polypeptide of the invention sH1 mini-HAGW1.5E2-FFH (SEQ ID NO: 158) adjuvated with 10 μg Matrix-M. As apositive control for the challenge model CR6261 (15 mg/kg) wasadministered 1 day prior to challenge (n=8), while 3 immunizations withPBS served as a negative control (n=16). Four weeks after the lastimmunization mice were challenged with 25×LD50 challenge virus andmonitored (survival, weight, clinical scores) for 3 weeks.

To verify immunogenicity polypeptide of the invention sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158), pre-challenge sera (day −1) were tested inELISA assay for binding to FL HA from H1N1 A/Brisbane/59/07. Todetermine whether mini-HA induced antibodies bind at close proximity tothe CR9114 epitope, a CR9114 competition ELISA was performed.Competition data were visualized as ‘% competition’, defined as(A−P)/A×100), where A is the maximum OD signal of CR9114 binding to FLHA when no serum is present and P is the OD signal of CR9114 binding toFL HA in presence of serum at a given dilution or expressed using theslope OD metric to be able to quantify responses; for reference CR9114and CR8020 (starting concentration 5 μg/ml) solutions were included.

Results

-   -   Experiment is valid; all mice in the PBS control group (n=16)        succumb to infection at or before day 9 post challenge (median 8        days), whereas the positive control group (n=8, 15 mg/kg CR6261,        1 day before challenge) is fully protected (p<0.001).    -   Three immunizations polypeptide of the invention sH mini-HA        GW1.5E2-FFH (SEQ ID NO: 158), adjuvated with Matrix-M lead to        significant increase in survival proportion (p<0.001), increase        in survival time (p<0.001), decrease in bodyweight loss        (p<0.001) and reduction in clinical score (p<0.001), compared to        the PBS control group (FIG. 35).    -   Pre-challenge IgG antibody titers to H1N1 A/Brisbane/59/07 FL HA        induced by polypeptide of the invention sH1 mini-HA GW1.5E2-FFH        (SEQ ID NO: 158) are significantly higher compared to PBS        (p<0.001) (FIG. 36A).    -   Matrix-M adjuvated polypeptide of the invention sH1 mini-HA        GW1.5E2-FFH (SEQ ID NO: 158) induce significantly higher CR9114        competition titers compared to PBS (p<0.001) (FIG. 36B).

CONCLUSION

Matrix-M adjuvated polypeptide of the invention sH1 mini-HA GW 1.5E2-FFH(SEQ ID NO: 158) confers protection against lethal challenge with H1N1A/Puerto Rico/8/34.

TABLE 1 Standard amino acids, abbreviations and properties Side chainSide chain Amino Acid 3-Letter 1-Letter polarity charge (pH 7.4) alanineAla A nonpolar Neutral arginine Arg R polar Positive asparagine Asn Npolar Neutral aspartic acid Asp D polar Negative cysteine Cys C nonpolarNeutral glutamic acid Glu E polar Negative glutamine Gln Q polar Neutralglycine Gly G nonpolar Neutral histidine His H polar positive (10%)neutral (90%) isoleucine Ile I nonpolar Neutral leucine Leu L nonpolarNeutral lysine Lys K polar Positive methionine Met M nonpolar Neutralphenylalanine Phe F nonpolar Neutral proline Pro P nonpolar Neutralserine Ser S polar Neutral threonine Thr T polar Neutral tryptophan TrpW nonpolar Neutral tyrosine Tyr Y polar Neutral valine Val V nonpolarNeutral

TABLE 2Sequence alignment of H1 sequences according to particular embodiments ofthe invention 1. A/Solomon Islands/6/2003 (H1N1) (SEQ ID NO: 25)2. A/Brisbane/59/2007 (H1N1) (SEQ ID NO: 1)3. A/New Caledonia/20/1999 (HIN1) (SEQ ID NO: 26)4. A/California/07/2009 (H1N1) (SEQ ID NO: 27)5. A/swine/Hubei/S1/2009 (H1N1) (SEQ ID NO: 28)6. A/swine/Haseluenne/IDT2617/2003 (H1N1) (SEQ ID NO: 29)7. A/NewYork/8/2006 (H1N1) (SEQ ID NO: 30)8. A/SolomonIslands/3/2006 (H1N1) (SEQ ID NO: 31)9. A/NewYork/146/2000 (H1N1) (SEQ ID NO: 32)10. A/NewYork/653/1996 (H1N1) (SEQ ID NO: 33)11. A/Beijing/262/1995 (H1N1) (SEQ ID NO: 34) 12. A/Texas/36/1991 (H1N1)(SEQ ID NO: 35) 13. A/Singapore/6/1986 (H1N1) (SEQ ID NO: 36)14. A/Chile/1/1983 (H1N1) (SEQ ID NO: 37) 15. A/Baylor/11515/1982 (H1N1)(SEQ ID NO: 38) 16. A/Brazil/11/1978 (H1N1) (SEQ ID NO: 39)17. A/USSR/90/1977 (H1N1) (SEQ ID NO: 40) 18. A/NewJersey/8/1976 (H1N1)(SEQ ID NO: 41) 19. A/Denver/1957 (H1N1) (SEQ ID NO: 42)20. A/Albany/4835/1948 (H1N1) (SEQ ID NO: 43)21. A/FortMonmouth/1/1947 (H1N1) (SEQ ID NO: 44)22. A/Cameron/1946 (H1N1) (SEQ ID NO: 45) 23. A/Weiss/1943 (H1N1)(SEQ ID NO: 46) 24. A/Iowa/1943 (H1N1) (SEQ ID NO: 47)25. A/Bellamy/1942 (H1N1) (SEQ ID NO: 48) 26. A/PuertoRico/8/1934 (H1N1)(SEQ ID NO: 49) 27. A/WSN/1933 (H1N1) (SEQ ID NO: 50)28. A/SouthCarolina/1/1918 (H1N1) (SEQ ID NO: 51)  1.MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL  60 2. MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL ENSHNGKLCL 60  3.MKAKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL  60 4. MKAILVVLLY TFATANADTL CIGYEANNST DTVDTVLEKN VTVTHSVNLL EDKHNGKLCK 60  5.MEAKLFVLFC AFTALKADTF CVGYHANYST HTVDTILEKN VTVTHSVNLL ENSHNGKLCS  60 6. MEAKLFVLFC AFTALKADTI CVGYHANNST DTVDTILEKN VTVTHSINLL ENNHNGKLCS 60  7.MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL  60 8. MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60  9.MKAKLLVLLC AFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR  6010. MKAKLLVLLC AFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 11.MKAKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL  6012. MKAKLLVLLC AFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 13.MKAKLLVLLC AFTATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR  6014. MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDNENGKLCK 60 15.MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR  6016. MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 17.MKAKLLVLLC ALSATDADTI CIGYEANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR  6018. MKAKLLVLLC AFTATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 19.MKAKLLILLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR  6020. MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 21.MKAKLLILLC ALTATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR  6022. MKAKLLILLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 23.MKARLLVLLC ALAATDADTI CIGYHANNST DTVDTILEKN VTVTHSVNLL EDSHNGKLCR  6024. MKARLLVLLC ALAATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 25.MKARLLVLLC AIAATDADTI CIGYHANNST DTVDTILEKN VTVTHSVNLL EDSHNGKLCR  6026. MEANILVLLC ALAAADADTI CIGYHANNST DTVDTVLEKN VTVTHSVNIL EDSHNGKLCR 60 27.MKAKLLVILY AEVATDADTI CIGYHANNST DTVDTIFEKN VAVTHSVNLL EDRHNGKLCK  6028. MEARLLVLLC AFAATNADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCK 60 *:. *::**  :: :: ***: ********** *****::*** *:******** *: ******* 1. LKGIAPLQLG NCSVAGWILG NPECELLISR ESWSYIVEKP NPENGTCYPG HFADYEELRE120  2.LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVEKP NPENGTCYPG HFADYEELRE 120 3. LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVETP NPENGTCYPG YFADYEELRE120  4.LRGVAPLHLG KCNIAGWILG NPECESLSTA SSWSYIVETP SSDNGTCYPG DFIDYEELRE 120 5. LNGKIPLOLG NCNVAGWILG NPKCDLLLTA NSSSYIIETS KSKNGACYPG EFADYEELKE120  6.LNGKAPLQLG NCNVAGW1LG NPECDLLLTV DSWSYIIETS NSKNGACYPG EFADYEELRE 120 7. LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVETP NPENGTCYPG YFADYEELRE120  8.LKGIAPLQLG NCSVAGWILG NPECELLISR ESWSYIVEKT NPENGTCYPG HFADYEELRE 120 9. LKGTAPLQLG NCSIAGWILG NPECESLFSK ESWSYIAETP NPKNGTCYPG YFADYEELPE120 10.LKGTAPLQLG NCSVAGWILG NPECESLFSK ESWSYIAETP NPENGTCYPG YFADYEELRE 12011. LKGIAPLQLG NCSVAGWILG NPECESLISK ESWSYIVETP NPENGTCYPG YFADYEELRE120 12.LKGIAPLQLG NCSVAGWILG NPKCESLFSK ESWSYIAETP NPENGTCYPG YFADYEELRE 12013. LKGIAPLQLG NCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE120 14.LKGIAPLQLG KCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE 12015. LKGIAPLQLG KCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE120 16.LKGIAPLQLG KCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELPE 12017. LKGIAPLQLG KCNIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE120 18.LKGIAPLQLG NCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE 12019. LKGKAPLQLG NCNIAGWVIG NPECESLLSN RSWSYTAETP NSENGTCYPG DFADYEELRE120 20.LKGIAPLQLG KCNIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE 12021. LKGIAPLQLG KCNIAGWILG NPECESLLSK RSWSYIAETP NSENGACYPG DFADYEELRE120 22.LKGIAPLQLG KCNIAGWILG NPECESLLSK RSWSYTAETP NSENGACYPG DFADYEELRE 12023. LKGIAPLQLG KCNIAGWILG NPECESLLSE RSWSYIVEIP NSENGTCYPG DFTDYEELRE120 24.LKGIAPLQLG KCNIAGWILG NPECESLLSE RSWSYIVETP NSENGTCYPG DFIDYEELRE 12025. LKGIAPLQLG KCNIAGWILG NPECESLLSE RSWSYIVETP NSENGTCYPG DFIDYEELRE120 26.LKGIAPLQLG KCNIAGWLLG NPECDPLLPV RSWSYIVETP NSENGICYPG DFIDYEELRE 12027. LKGIAPLQLG KCNITGWLLG NPECDSLLPA RSWSYIVETP NSENGACYPG DFIDYEELRE120 28.LKGIAPLQLG KCNIAGWLLG NPECDLLLTA SSWSYIVETS NSENGTCYPG DFIDYEELRE 120*:* ***:** :*.::**:** **:*: * .   *****.* . ...** ****  * *******  1.QLSSVSSFER FEIFPKESSW PNHTTT-GVS ASCSHNGESS FYKNLLWLTG KNGLYPNLSK 179 2. QLSSVSSFER FEIFPKESSW PNHTVT-GVS ASCSHNGESS FYRNLLWLTG KNGLYPNLSK179  3.QLSSVSSFER FEIFPKESSW PNHTVT-GVS ASCSHNGKSS FYRNLLNLTG KNGLYPNLSK 179 4. QLSSVSSFER FEIFPKTSSW PNHDSNKGVT AACPHAGAKS FYKNLIWLVK KGNSYPKLSK180  5.DLSTVSSFER FEIFPKAISW PDHDATRGTT VACSHSGVNS FYRNLLSTVK KGNSYPKLSK 180 6. QLSTVSSFER FEIFTKATSW PNHDTTRGTT ISCSHSGANS FYRNLLWIVK KGNSYPKLSK180  7.QLSSVSSFER FEIFPKESSW PNHTVT-GVS ASCSHNGKSS FYRNLLWLTG KNGLYPNLSK 179 8. QLSSVSSFER FEIFPKESSW PNHTTT-GVS ASCSHNGESS FYKNLLWLTG KNGLYPNLSK179  9.QLSSVSSFER FEIFTKDSSW PNHTVTKGVT ASCSHNGKSS FYKNLLWLTE KNGLYPNLSK 18010. QLSSVSSFER FEIFPKESSW PNHTVTKGVT ASCSHNGKSS FYKNLLWLTE KNGLYPNLSK180 11.QLSSVSSFER FEIFPKESSW PNHTVT-GVT ASCSHNGKSS FYRNLLWLTE KNGLYPNLSN 17912. QLSSVSSFER FEIFPKESSW PNHTVTKGVT TSCSHNGKSS FYRNLLWLTK KNGLYPNVSK180 13.QLSSVSSFER FEIFPKESSW PNHTVTKGVT ASCSHKGRSS FYRNLLWLTK KNGSYPNLSK 18014. QLSSVSSFER FEIFPKESSW PKHNVTKGVT AACSHKGKSS FYRNLLWLTE KNGSYPNLSK180 15.QLSSVSSFER FEIFPKESSW PKHSVTRGVT ASCSHKGKSS FYRNLLWLTE KNGSYPNLSK 18016. QLSSVSSFER FEIFPKERSW PKHNITRGVT ASCSHKGKSS FYRNLLWLTE KNGSYPNLSK180 17.QLSSVSSFER FEIFPKERSW PKHNVTRGVT ASCSHKGKSS FYRNLLWLTE KNGSYPNLSK 18018. QLSSVSSFER FEIFPKESSW PNHTVTKGVT ASCSHKGRSS FYRNLLWLTK KNGSYPNLSK180 19.QLSSVSSFER FEIFPKERSW PNHTTR-GVT AACPHARKSS FYKNLVWLTE ANGSYPNLSR 17920. QLSSVSSFER FEIFPKERSW PKHNITRGVT AACSHKGKSS FYRNLLWLTE KNGSYPNLNK180 21.QLSSVSSFER FEIFPKERSW PKHNITRGVT AACSHAGKSS FYKNLLWLTE TDGSYPKLSK 18022. QLSSVSSFER FEIFPKGRSW PEHNIDIGVT AACSHAGKSS FYKNLLWLTE KDGSYPNLNK180 23.QLSSVSSFER FEIFPKESSW PKHNTARGVT AACSHAGKSS FYRNLLWLTE KDGSYPNLKN 18024. QLSSVSSFER FEIFSKESSW PKHTTG-GVT AACSHAGKSS FYRNLLWLTE KDGSYPNLNN179 25.QLSSVTSFER FEIFPKETSW PKHNTTKGVT AACSHAGKCS FYRNLLWLTE KDGSYPNLNN 18026. QLSSVSSFER FEIFPKESSW PNHNTN-GVT AACSHEGKSS FYRNLLWLTE KEGSYPKLKN179 27.QLSSVSSLER FEIFPKESSW PNHTFN-GVT VSCSHRGKSS FYRNLLWLTK KGDSYPKLTN 17928. QLSSVSSFEK FEIFPKTSSW PNHETTKGVT AACSYAGASS FYRNLLWLTK KGSSYTKLSK180 *****:*:*: ****.*  ** *:*    **: .:*.:    * **:**:**.    . **::.. 1. SYANNKEKEV LVLWGVHHPP NIGDQRAIYH KENAYVSVVS SHYSRKFTPE IAKRPKVRDQ239  2.SYANNKEKEV LVLWGVHHPP NIGNQKAIYH TENAYVSVVS SHYSRKFTPE IAKRPKVRDQ 239 3. SYVNNKEKEV LVLWGVHHPP NIGNQRALYM TENAYVSVVS SHYSRRFTPE IAKRPKVRDQ239  4.SYINDKGKEV LVIWGIHHPS TSADQQSLYQ NADATIEVGS SRYSKKFKPE LAIRPKVRXX 240 5. SYTNNKGKEV LVIWGVHHPP TDSVQQTLYQ NKHTYVSVGS SKYYKRFTPE IVARPKVRGO240  6.SYTNNKGKEV LVIWGVHHPP TDSDQQTLYQ NNHTYVSVGS SKYYQRFTPE IVTRPKVRGQ 240 7. SYANNKEKEV LVLWGVHHPP NIGDQRALYH TENAYVSVVS SHYSRRFTPE LAKPPKVRDQ239  8.SYANNKEKEV LVLWGVHHPP NIGDQRALYH KENAYVSVVS SHYSRKFTPE IAKRPKVRDQ 239 9. SYVNKKGKEV LVLWGVHHPS NMGDQRAIYH KENAYVSVLS SHYSRPFTPF IAKRPKVRDQ240 10.SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSVVS SHYSRRFTPE ITKPPKVRDQ 24011. SYVNNKEKEV LVLWGVHHPS NIRDQRAIYH TENAYVSVVS SHYSRRFTPE LAKRPKVRGO239 12.SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSVVS SHYSRPFTPF IAKRPKVRDQ 24013. SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSVVS SHYNRRFTPE IAKRPKVRDQ240 14.SYVNNKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSVVS SHYNRRFTPE LAKRPKVPNO 24015. SYVNDKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSVVS SHYNRRFTPE IAKRPKVRDQ240 16.SYVNNKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSVVS SNYNRRFTPE LAKRPKVRGQ 24017. SYVNNKEKEV LVLWGVHHPS NIEDQKTIYP KENAYVSVVS SNYNRRFTPE LAERPKVRGQ240 18.SYVNNKEKEV LVLWGVHHPS NIGDORAIYH TENAYVSVVN SHYNRRFTPE IAKRPKVRDQ 24019. SYVNNQEKEV LVLWGVHHPS NIEEQRALYR KDNAYVSVVS SNYNRRFTPE IAKRPKVRDQ239 20.SYVNNKEKEV LVLWGVHHPS NIEDQKTLYR KENAYVSVVS SNYNRRFTPE LAEPPKVRGQ 24021. SYVNNKEKEV LVLWGVHHPS NIEDQKTLYR KENAYVSVVS SNYNRRFTPE LAERPKVRGQ240 22.SYVNKKEKEV LILWGVHHPP NIENQKTLYR KENAYVSVVS SNYNRRFTPE LAERPKVRGQ 24023. SYVNKKGKEV LVLWGVHHPS SIKEQQTLYQ KENAYVSVVS SNYNRRFTPE LAEPPKVRDQ240 24.SYVNKKGKEV LVLWGVHHPS NIKDQQTLYQ KENAYVSVVS SNYNRRFTPE LAERPKVRGO 23925. SYVNKKGKEV LVLWGVHHPS NIKDQQTLYQ KENAYVSVVS SNYNRPFTPF LAERPKVRGQ240 26.SYVNKKGKEV LVIWGIHHPP NSKEQQNLYQ NENAYVSVVT SNYNRRFTPE LAEPPKVRDQ 23927. SYVNNKGKEV LVLWGVHHPS SSDEQQSLYS NGNAYVSVAS SNYNRRFTPE LAARPKVKDO239 28.SYVNNKGKEV LVLWGVHHPP TGTDQQSLYQ NADAYVSVGS SKYNRRETTE IAARPKVRDQ 240** *.: *** *:***:***. .  :*: :*  . :*** * : *.*.::*.** *: ****:  1.EGRINYYWTL LEPGDTIIFE ANGNLIAPRY AFALSRGFGS GIINSNAPMD ECDAKCQTPQ 299 2. EGRTNYYWTL LEPGDTIIFE ANGNLIAPRY AFALSRGFGS GIINSNAPMD KCDAKCQTPQ299  3.EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNAPMD ECDAKCQTPQ 299 4. EGRMNYYWTL VEPGDKITFE ATGNLVVPRY AFAMEPNAGS GIIISDTPVH DCNTTCQTPK300  5.AGRMNYYNTL FDQGDTITFE ATGNLIAPWH AFALKKGSSS GIMLSDAQVH NCTTKCQTPH 300 6. AGRMNYYWTL LDQGDTITFE ATGNLIAPWH AFALNKGPSS GIMISDAHVH NCTTKCQTPH300  7.EGRINYYWTL LEPGDTIIFE ANGNIIATRF AFALSRGFGS GIITSNATMD ECDAKCQTPQ 299 8. EGRINYYWTL LEPGDTIIFE ANGNLIAPRY AFALSRGFGS GIINSNAPMD ECDAKCQTPQ299  9.EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIIISNASMG ECDAKCQTPQ 30010. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMG ECDAKCQTPQ300 11.EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNAPMN ECLAKCQTPQ 29912. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMD ECDAKCQTPQ300 13.EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMD ECDAKCQTPQ 30014. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMD ECDAKCQTPQ300 15.EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GITTSNVSMD ECDAKCQTPQ 30016. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMD ECDTKCQTPQ300 17.AGRINYYWTL LEPGDTIIFE ANGNLIAPWH AEALNRGEGS GIITSNASMD ECDTKCQTPQ 30018. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMD ECDAKCQTPQ300 19.SGRMNYYWTL LEPGDTIIFE ATGNLIARNY AFALSRGPGS GIITSNAPLD ECDTKCQTPQ 29920. AGRINYYWTL LEPGDTIIFE ANGNLIAPWH AFALSRGFGS GIITSNASMD ECDTKCQTPQ300 21.AGRINYYNTL LEPGDTIIFE ANGNLIAPWY AFALSRDEGS GIITSNASMD ECDTKCQTPQ 30022. AGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALNRGIGS GIITSNASMD ECDTKCQTPQ300 23.AGRMNYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASME ECDTKCQTPQ 30024. AGRINYYWTL LKPGDTIMFE ANGNLIAPWY AFALSRGFGS GIITSNASMH ECDTKCQTPQ299 25.AGRMNYYWTL LEPGDTIIFE ANGNIIATWY AFALSRGFGS GIITSNASME ECNTKCQTPQ 30026. AGRMNYYWTL LKPGDTIIFE ANGNLIAPMY AFALPRGEGS GIITSNASME ECNTKCQTPL299 27.HGRMNYYWTL LEPGDTIIFE ATGNLIAPWY AFALSRGFES GIITSNASMH ECNTKCQTPQ 29928. AGRMNYYWTL LEPGDTITFE ATGNLIATWY AFALNRGSGS GIITSDATVE DCNTKCQTPH300  **:****** ::***.* ** *.***:.* . ***: *.  * *** *:..:  .*::.****  1. GAINSSLPFQ NVHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG359  2.GAINSSLPFQ NVEPVTIGEC PKYVPSAKLP MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359 3. GAINSSLPFQ NVHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG359  4.GAINTSLPFQ NIHPITIGKC PKYVKSTKLR LATGLPNIPS IQSRGLFGAI AGFIEGGWTG 360 5. GALKNNLPLQ NVELFTIGEC PKYVKSTQLR MATGLRNIPS IQSRGLFGAI AGFIEGGRTG360  6.GALKSNLPFQ NVHPSTIGEC PKYVKSTQLR MATGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 7. GAINSSLPFQ NVHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG359  8.GAINSSLPFQ NVHPVTIGEC PKYVPSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359 9. GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNVPS IQSRGLFGAI AGFIEGGWTG360 10.GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 36011. GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG359 12.GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 36013. GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG350 14.GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 36015. GAINSSLPFQ NVEPVTIGEC PKYVPSTKLP MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG360 16.GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 36017. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG360 18.GAINSSLPFQ NVHPVTIGEC PKYVPSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 36019. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS VQSRGLFGAI AGFIEGGWTG359 20.GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 36021. GAINSSLPFQ NIHPVTIGEC PKYVKSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG360 22.GAINSSLPFQ NIHPFTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWDG 36023. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG360 24.GAINSSLPFQ NIHPVTIGEC PKYVPSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 35925. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG360 26.GAINSSLPYQ NIHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 35927. GSINSNLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQYRGLFGAI AGFIEGGWTG359 28.GAINSSLPFQ NIHPVTIGEC PKYVPSTKLP MATGLRNIPS IQSRGLFGAI AGFIEGGWTG 360*:**:.**:* *:**.***:* ****:*:*** :.*****:** :* ******* ******** *  1.MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GI T NKVNSVI EKMNTQFTAV GKEFNKLERR 419 2. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GI T NKVNSVI EKMNTQFTAV GKEFNKLERR419  3. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GI TNKVNSVI EKMNTQFTAV GKEFNKLERR 419  4.MVDGWYGYHH QNEQGSGYAA DLKSTQNAID EI T NKVNSVI EKMNTQFTAV GKEFNHLEKR 420 5. MIDGWYGYHH QNEQGSGYAA DQKSTQIAID GI N NKANSVI GKMNIQLTSV GKEFNSLEKR420  6. MIDGWYGYHH QNEQGSGYAA DQKSTQIAID GI NNKVNSII EKMNTQFTSV GKEFNDLEKR 420  7.MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419 8. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR419  9.MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSII EKMNTQFTAV GKEFNKLEKR 42010. MIDGWYGYHH QNEQGSGYAA DQKSTQNAID GITNKVNSVI EKMNTQFTAV GKEFNKLERR420 11.MMDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKIERR 41912. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR420 13.MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKIETR 42014. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSII EKMNTQFTAV GKEFNKLEKR420 15.MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR 42016. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR420 17.MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR 42018. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR420 19.MMDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR 41920. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR420 21.MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN WITNKVNSVI EKMNTQFTAV GKEFNKLERR 42022. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR420 23.MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEENNLEKR 42024. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNNLEKR419 25.MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNNLEKR 42026. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNIQFTAV GKEFNKIEKR419 27.MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFTNLEKR 41928. MIDGWYGYHH QNEQGSGYAA DQKSTQNAID GITNKVNSVI EKMNTQFTAV GKEFNNLERR420 *:******** ********** * *******:  *******:* **** ***** *****:**:* 1. MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSOL KNNAKEIGNG479  2.MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479 3. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG479  4.IENLNKKVDD GFLDIWTYNA ELLVLLENEP TLDYHDSNVK NLYEKVRSQL KNNAKEIGNG 480 5. KENLNKTVDD RFLDVWTFNA ELLVLLENQR TLEFHDLNIK SLYEKVKSHL RNNDKEIGNG480  6.IENLNKKVDD GFLDVWTYNA ELLILLENER TLDFHDFNVK NLYEKVKSQL RNNAKEIGNG 480 7. MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG479  8.MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479 9. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDLNVK NLYEKVKNQL KNNAKEIGNG480 10.MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKTQL KNNAKEIGNG 48011. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSOL KNNAKEIGNG479 12.MENLNKKVDD GFLDIWTYNA ELLVLLENGR TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 48013. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG480 14.MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSOL KNNAKEIGNG 48015. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG480 16.MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 48017. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG480 18.MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 48019. MENLNKKVDD GFMDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKNQL RNNAKELGNG479 20.MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 48021. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKNQL RNNAKEIGNG480 22.MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKNQL RNNAKEIGNG 48023. MENLNKKVDD GFIDIWTYNA ELLILLENER TLDFHDSNVK NLYEKVKSQL RNNAKEIGNG480 24.MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKNOL RNNAKEIGNG 47925. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL RNNAKEIGNG480 26.MENLNNKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 47927. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDLNVK NLYEKVKSOL KNNAKEIGNG479 28.IENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFEDSNVR NLYEKVKSQL KNNAKEIGNG 480:****:**** **:******* ***:**** * ***:** **: ******:.** :*****:***  1.CFEFYHKCND ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 539 2. CFEFYHKCND ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS539  3.CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 539 4. CFEFYHKCDN TCMESVKNGT YDYPKYSEEA KLNREEIDGV KLESTRIYQI LAIYSTVASS540  5.CFEFYHKRDN ECLECVKNGT YNYPKYSEES KFNREEIVGV KLESMGIHQI LAIYSTVASS 540 6. CFEFYHKCDN ECMESVKNGT YNYPKYSEES KLNREKIDGV KLESMGVHQI LAIYSTVASS540  7.CFEFYHKCND ECMESVKNGT YDYPKYSEES KLNRERIDGV KLESMGVYQI LAIYSTVASS 539 8. CFEFYEKCND ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS539  9.CFEFYHKCNN ECMESVKNGT YDYPKYSKES KLNREKIDGV KLESMGVYQI LAIYSTVASS 54010. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS540 11.CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 53912. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNRGKIDGV KLESMGVYQI LAIYSTVASS540 13.CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 54014. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS540 15.CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 54016. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS540 17.CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 54018. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS540 19.CFEFYHKCDN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYRI LAIYSTVASS 53920. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS540 21.CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 54022. CFEFYHKCNN ECMESVKNGT YDYPKFSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS540 23.CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 54024. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTAASS539 25.CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 54026. CFEFYHKCDN ECMESVPNGT YDYPKYSEES KLNREKVDGV KLESMGIYQI LAIYSTVASS539 27.CFEFYHKCDN ECMESVRNGT YDYPKYSEES KINREKIDGV KLESMGVYQI LAIYSTVASS 53928. CFEFYHKCDD ACMESVRNGT YDYPKYSEES KLNREEIDGV KLESMGVYQI LAIYSTVASS540 ********::  *****:*** *****:*:*: **** .:*** ****  :*:* ******.*** 1. LVLLVSLGAI SFWMCSNGSL QCRICI 565  2. LVLLVSLGAI SFWMCSNGSL QCRICI565  3. LVLLVSLGAI SFWMCSNGSL QCRICI 565  4.LVLVVSLGAI SFWMCSNGSL QCRICI 566  5. LVLLVSLGAI SFWMCSNGSL QCRVCI 566 6. LVLLVSLGAI SFWMCSNGSL QCRICI 566  7. LVLLVSLGAI SFWMCSNGSL QCRICI565  8. LVLLVSLGAI SFWMCSNGSL QCRICI 565  9.LVLLVSLGAI SFWMCSNGSL QCRICI 566 10. LVLLVSLGAI SFWMCSNGSL QCRICI 56611. LVLLVSLGAI SFWMCSNGSL QCRICI 565 12. LVLLVSLGAI SFWMCSNGSL QCRICI566 13. LVLLVSLGAI SFWMCSNGSL QCRICI 566 14.LVLLVSLGAI SFWMCSNGSL QCRICI 566 15. LVLLVSLGAI SFWMCSNGSL QCRICI 56616. LVLLVSLGAI SFWMCSNGSL QCRICI 566 17. LVLLVSLGAI SFWMCSNGSL QCRICI566 18. LVLLVSLGAI SFWMCSNGSL QCRICI 566 19.LVLLVSLGAI SFWMCSNGSL QCRICI 565 20. LVLLVSLGAI SFWMCSNGSL QCRICI 56621. LVLLVSLGAI SFWMCSNGSL QCRICI 566 22. LVLLVSLGAI SFWMCSNGSL QCRICI566 23. LVLLVSLGAI SFWMCSNGSL QCRICI 566 24.LVLLVSLGAI SFWMCSNGSL QCRICI 565 25. LVLLVSLGAI SFWMCSNGSL QCRICI 56626. LVLLVSLGAI SFWMCSNGSL QCRICI 565 27. LVLLVSLGAI SFWMCSNGSL QCRICI565 28. LVLLVSLGAI SFWMCSNGSL QCRICI 566 ***:****** ********** ******

TABLE 3 Polypeptides expressed in P. pastoris. Expression and CR6261binding were determined as described and the ratio of binding andexpression signals calculated. SET1 fold increase Fusion peptide areaB-loop CR261 of ratio over 337 340 352 353 402 406 409 413 416 bindingHTRF parental H1 E, I, I, K, D, F, I, K, E, K, F, I, N, S, A, G, I, R,F, I, N, S H, I, L, N, clone signal signal ratio mini-HA K, V R, T V, YR, T M, V T, Y T, V T, Y R, S 239E11 1076944 1492 721.81 121.52 K I Y TM F I N R 127H1 800024 6572 121.73 20.49 K K F T M Y I Y S 171E5 87970411508 76.44 12.87 K T F T M I A F S 239D2 570424 9279 61.47 10.35 K K FT M I V F N 247B2 414984 7583 54.73 9.21 K I Y T V Y I F S 253D4 3958247546 52.45 8.83 K T F T M Y A Y H 252F5 421824 8621 48.93 8.24 V K Y T MY V Y N 220C9 1086064 22606 48.04 8.09 K T F T M F T Y L 125D3 1398242937 47.61 8.02 K K F T M Y G T H 137C11 416504 9167 45.44 7.65 V K F TM Y I N H 131B5 844344 20419 41.35 6.96 K T F T M I V Y H 233F11 58302414389 40.52 6.82 K K Y T M T I G S 234C5 377864 9465 39.92 6.72 I I Y TM F T N L 115A1 1176904 30389 38.73 6.52 K K V T M I V Y I 185G7 50586413560 37.31 6.28 K K Y T M I V I S 275D4 327344 9030 36.25 6.10 K K Y TM T T S S 244B8 273744 7757 35.29 5.94 I T Y T M Y A I S 252B8 2849848252 34.54 5.81 K I Y T M S I N L 213C11 667024 20624 32.34 5.44 V K Y TM I V F H 174G3 491184 15320 32.06 5.40 K T Y K V S G Y L 125D10 1339044241 31.57 5.31 K I Y T M Y V N R 127A7 233064 7498 31.08 5.23 E T Y T MI I I L 304G11 110504 3588 30.8 5.19 K K Y K M F T F S 162A11 36402411939 30.49 5.13 V K Y T M F A F I 271F10 315304 10348 30.47 5.13 I K YT M I A I L 218G11 958504 33710 28.43 4.79 I T Y I M I I I N 251C8269544 9634 27.98 4.71 K T Y K M Y I N L 258A6 165624 6004 27.59 4.64 IT Y T M Y T F H 134A4 456304 17366 26.28 4.42 K I Y I M I A Y N 214C11317904 12120 26.23 4.42 E I Y T M Y V S S 182G8 399864 15262 26.2 4.41 KK Y T M T V I I 113E7 966064 38018 25.41 4.28 K K F T M Y T I H 230G9854584 34093 25.07 4.22 K K Y T M Y T F R 222G4 419064 16996 24.66 4.15K T F I V I I Y L 182D7 418944 17096 24.51 4.13 I T Y T M I I F N 272H2263264 10844 24.28 4.09 K T Y T M S A N H 191C8 309064 12753 24.23 4.08I T Y T V I A F I 123C10 237824 9843 24.16 4.07 K I Y K M F A T L 284B91663504 70812 23.49 3.95 K T Y R M I R T L 134A3 531784 23414 22.71 3.82K K F I M I I N S 188F4 287384 12888 22.3 3.75 K K Y T M S V T H 189B7336344 15207 22.12 3.72 E T F T M Y V F N 148D5 329144 14994 21.95 3.70E T Y I M F G S H 194C8 242304 11113 21.8 3.67 I T F T M F V F I 188A8279144 13001 21.47 3.61 K T Y K M F V S I 162B3 279584 13159 21.25 3.58V T Y T M Y T N N 204C5 832784 39330 21.17 3.56 V K F T V I I Y L 216E5334904 15873 21.1 3.55 V T F T M F R Y R 129C2 199464 9486 21.03 3.54 VR Y I M I I Y S 286E8 158704 7662 20.71 3.49 E I F T M F I Y S 264G4180504 8751 20.63 3.47 K R Y T V I V F S 214C4 302264 14709 20.55 3.46 II F T V F A S S 125A8 212224 10327 20.55 3.46 K I F T V I V Y I 123G2498584 24442 20.4 3.43 I T Y I M Y T F L 187C6 345464 16932 20.4 3.43 EK Y K M F I I H 134H10 591704 29253 20.23 3.41 K T Y T V I T F I 187H10299224 15289 19.57 3.29 K T Y I M I G F L 101D4 336584 17243 19.52 3.29I K Y I M I I S N 193B6 206904 10650 19.43 3.27 K K Y R M F I S N 137C5295944 15406 19.21 3.23 I R F T V I I N N 112F3 449824 24169 18.61 3.13V R F I M I I Y S 176A5 193104 10476 18.43 3.10 I T F T V F I F I 213B2131704 7178 18.35 3.09 K K Y T M T V F L 307A10 114984 6348 18.11 3.05 IK F T M Y G Y H 126C3 219944 12413 17.72 2.98 E T F I M F G T I 263B6151184 8800 17.18 2.89 I T Y I M S T Y I 138F11 147864 8788 16.83 2.83 ER Y R M F V F L 134D3 303504 18129 16.74 2.82 E R F I M Y T F S 131D5344504 20857 16.52 2.78 V T Y I V I A F S 138F8 347704 21081 16.49 2.78K T Y I M Y A F H 301F11 116904 7108 16.45 2.77 V T F T V Y I S H 112G6543944 33149 16.41 2.76 V R Y I M F I S I 245C9 180024 10980 16.4 2.76 VR F T V F V T L 123E2 477064 29184 16.35 2.75 V T Y T V F V F S 266A1190584 5696 15.9 2.68 V T Y T M Y I T R 104C4 521224 34458 15.13 2.55 V KY I M F G F N 194E4 408584 27424 14.9 2.51 E K F T M I T F I 206B11358744 24697 14.53 2.45 V R Y T M F T I L 192C4 343184 23932 14.34 2.41K T Y K M I V T N 125H3 317384 22785 13.93 2.35 I T F T M I A Y R 145C9182344 13108 13.91 2.34 I T F I V Y I S N 243D6 132144 9596 13.77 2.32 IR F T M N V Y R 182D3 142664 10487 13.6 2.29 I T Y R M F A G S 181H9310504 23153 13.41 2.26 V K F I M F V F N 163E3 183544 14033 13.08 2.20E K Y K M I V I L 145E7 132224 10312 12.82 2.16 I T F K V I I F S 275G3115104 9180 12.54 2.11 V T Y I M T A S S 191D5 123824 10048 12.32 2.07 IR F T M T G F S 188G10 142504 11593 12.29 2.07 V T Y I V I A F S 171F6140464 11555 12.16 2.05 K T Y T M S T Y L 125C2 83624 7009 11.93 2.01 II F T V I T S S 206B8 285824 24166 11.83 1.99 V I Y T M I T F H 145F2498504 42457 11.74 1.98 I K F T M F R F S 199F3 328504 29850 11.01 1.85K T Y T M N G S S 181H11 186664 17205 10.85 1.83 V T Y T M I I N R 188C8113344 10520 10.77 1.81 I K Y T M S T Y L 189E10 188864 18252 10.35 1.74K T Y T M S G S S 146G7 533864 52422 10.18 1.71 V T Y I M Y T T I 182H2109624 10976 9.99 1.68 K I F T V I I T L 262B9 94744 9584 9.89 1.66 I KY T M F R F R 145E8 211504 21732 9.73 1.64 E K F K V I V F I 249B11145184 14995 9.68 1.63 K K F T M S T G H 182C6 92944 9939 9.35 1.57 K RD I M F I N N SEQ ID NO: 6 AV + 2SD 9.28 1.56 SEQ ID NO: 6 AV 23807740100 5.94 1.00

TABLE 4 Polypeptides expressed in P. pastoris. Expression and CR6261binding were determined as described and the ratio of binding andexpression signals calculated. fold increase of ratio Set 2 over Fusionpeptide area B-loop CR6261 parental 337 340 352 353 406 409 413 416binding HTRF SEQ ID A, E, I, F, I, N, A, D, F, I, N, E, G, I, 402 F, H,F, I, E, K, I, L, clone signal signal ratio NO: 6 K, T, V S, T, Y S, T,V, Y K, R, V M, R, T L, Y S, T M, V R, S 86B4 1077144 13862 77.7 13.08 KN Y K M F I M I 7A7 987824 13452 73.43 12.36 T N Y V M Y F E R 55G7616184 8767 70.28 11.83 K N Y V M Y I M L 71H2 1109984 16750 66.27 11.16K N F K M L I V S 86B3 900904 14448 62.35 10.50 K N Y K M L I V R 71A41064144 17597 60.47 10.18 T N Y V M Y F E R 51G3 460304 7773 59.22 9.97T I F V M L F E S 84B8 582144 10091 57.69 9.71 K N Y I M F F M S 79C2364184 7116 51.18 8.62 T N Y R M F T V S 69G8 481344 9479 50.78 8.55 I NF R M L I V L 79D5 702584 13981 50.25 8.46 A N F K M L F V L 54H4 2917445857 49.81 8.39 K I Y K M L I E L 11H6 427384 9146 46.73 7.87 K N Y E MF T E S 90A9 413664 9025 45.84 7.72 K S Y V M Y T V S 75G5 1011384 2669537.89 6.38 E S Y V M L F E R 8A10 360104 9630 37.39 6.29 K N Y V M L I VR 72D4 329944 8881 37.15 6.25 V N F R M F S M S 74H9 1283144 35494 36.156.09 K N F K M Y F M S 88C5 471424 13355 35.3 5.94 K N Y R M L I V R61A9 383064 10864 35.26 5.94 T N F R M F F E L 86H9 457344 13340 34.285.77 K N F G M F T V S 71D3 1573024 46711 33.68 5.67 I S Y V M F I V L9C6 270984 8235 32.91 5.54 K T Y V M Y T K I 81F11 317824 9964 31.9 5.37K I F V M F F V S 84E10 255064 7996 31.9 5.37 I N F R M F S V S 71C41350144 44339 30.45 5.13 K N F G M F I V S 84D3 84424 2920 28.91 4.87 EN F K M L I E S 96H8 205904 7224 28.5 4.80 K Y Y K M F I M S 85A7 2357048416 28.01 4.72 K N Y E M L F V R 50G10 264144 9470 27.89 4.70 T N F E MF F V S 6A1 299824 10912 27.48 4.63 A N F R M F F M S 91C4 1157424 4483725.81 4.35 K N F G M L I M R 2C4 258264 10139 25.47 4.29 I N F V M F I VL 63C3 188184 7625 24.68 4.15 E T Y K M L F V L 850 196024 8115 24.164.07 K N V G M F F V I 67C10 306104 12907 23.72 3.99 E T F V M F F M L10F9 165984 7113 23.34 3.93 I I Y V M Y F E R 4C1 385504 16548 23.3 3.92K N S V M F I E I 86G3 183944 7995 23.01 3.87 T S Y V M F T V L 51G10215264 9727 22.13 3.73 A N Y R M F I K S 58A5 90744 4142 21.91 3.69 V TF R M L I M S 56F8 235344 10823 21.74 3.65 I N F E M F T E L 67C11209184 9856 21.22 3.57 K Y Y I M F F E I 91C8 333584 16012 20.83 3.51 KN F G M L I K S 48B11 302864 14946 20.26 3.41 I N A G M L I E S 78F1184104 4155 20.24 3.41 I I F R M Y F E I 76A10 136984 6841 20.02 3.37 I YF V M Y F E I 55H2 58104 2984 19.47 3.28 I 1 Y V M F F V S 74D7 35878418453 19.44 3.27 K N A G M F I M S 11B4 166464 8679 19.18 3.23 T S F V MY T V S 56F4 185984 9740 19.09 3.21 T T F E M F S M S 71E7 202704 1068818.97 3.19 K N S R M Y I E S 48B10 102904 5480 18.78 3.16 I F F K M L FM S 48D11 120584 5807 17.71 2.98 E Y Y V M F T V S 35H3 106224 609217.44 2.94 V S F V M L S M R 53G10 107784 6188 17.42 2.93 T N F V M L TV S 86F1 158624 9145 17.35 2.92 I I F V M Y I V I 9C10 114144 6595 17.312.91 I I Y V M H S V S 6E12 372504 22044 16.9 2.85 E N F I M L F V L 2D9316024 19245 16.42 2.76 K N N I M Y F E L 27B10 187344 11465 16.34 2.75K N N V M L F E S 79F8 185264 11801 15.7 2.64 I N V I M F T E S 11F4150824 9996 15.09 2.54 I Y F V M Y F V L 60A2 92664 6166 15.03 2.53 E NY V M F S E L 58C8 277144 18603 14.9 2.51 A S Y I M L S E L 12C6 28918420023 14.44 2.43 I N S V M L I E L 89F11 84824 5908 14.36 2.42 T I Y I ML S V S 96G5 108264 7589 14.27 2.40 V N F I M Y F M S 29C2 177904 1292113.77 2.32 K N F G M Y F M R 56D2 145624 10658 13.66 2.30 E T F I M F FK S 66C8 184544 13591 13.58 2.29 K N V I M L F V L 69D2 445704 3426613.01 2.19 V F F V M Y T E S 75E9 134504 10422 12.91 2.17 I I F G M F SE I 97G10 253104 20061 12.62 2.12 E S F I M F F E I 36E4 196104 1591712.32 2.07 I N N K M F F V L 7D9 77824 6320 12.31 2.07 K N F V M F F M L1F2 148544 12244 12.13 2.04 K N Y V M F F M I 76D10 113664 9729 11.681.97 T N A K M L T E S 36H2 171144 14761 11.59 1.95 T N Y K M H F M R86G2 69704 6069 11.49 1.93 E N F V M L I E R 63D3 145784 13100 11.131.87 K N I G M F T E L 96A7 83304 7575 11 1.85 V I F V M F S V S 36D671304 6569 10.85 1.83 I N A G M F T E I 91F10 14784 1394 10.6 1.78 T N YG M F I E R 80F10 90864 8609 10.55 1.78 I S V V M L I E S 75H8 10330410074 10.25 1.73 A N N V M F F M S 57B8 58384 5800 10.07 1.70 K I Y I MF F V I 8D7 73424 7324 10.03 1.69 K N F V M L F E L 58A11 53264 53639.93 1.67 V T Y I M F T V S 7B6 60384 6137 9.84 1.66 K I S E M F I M S87H5 78104 7994 9.77 1.64 E I F I M F F V S 70F6 418624 43334 9.65 1.63K N I G M L T E R 26H1 79744 8268 9.64 1.62 E N F I M L S V I 78G2 567046055 9.36 1.58 V I Y G M L F E S SEQ ID NO: 6 AV + 2SD 9.28 1.56 SEQ IDNO 238077 40100 5.94 1.00

TABLE 5 Polypeptides expressed in HEK293F. Expression and CR6261 bindingwere determined as described and the ratio of binding and expressionsignals calculated. The mutations included in each clone are indicatedin Table 4 and 5. fold increase of CR6261 ratio over binding HTRFparental SEQ ID Clone signal signal ratio NO: 6 127H1 24150000 32736373.77 4.25 86B4 19970680 334887 59.63 3.44 171E5 6625080 235511 28.131.62 7A7 6191080 242461 25.53 1.47 71H2 21080360 336346 62.67 3.61 220C98493560 162872 52.15 3.00 131B5 5725640 139561 41.03 2.36 115A1 9557640175377 54.50 3.14 74H9 26144240 344988 75.78 4.37 71C4 6413600 21449529.90 1.72 91C4 8442400 245138 34.44 1.98 113E7 13005960 260748 49.882.87 6E12 15326000 309443 49.53 2.85 181H9 11892520 324690 36.63 2.11SEQ ID NO: 6 AV 5661550 326077 17.36 1.00

TABLE 6 Naturally occuring sequence variation at the indicated positionsin % of total number of sequences for each subtype Position amino acidH1 H3 H5 H7 337 V 67 99 19 100 I 32 1 2 T 0.8 3 S 73 Y 0.1 N 0.5 A 2 G0.1 340 I 99 21 98 V 0.43 T 0.03 0.5 K 97 R 2 47 G 29 E 0.3 S 2 352 F100 100 100 100 353 I 99.9 100 100 100 L 0.1 402 M 100 100 T 99.8 100 S0.02

TABLE 7 Purification and strength of mAb binding of polypeptides VolumeYield Purity K_(d) ^(app) K_(d) ^(app) SEQ ID supernatant (mg/l of fromHP- CR6261 CR9114 NO: (ml) culture) SEC (%) (nM) (nM) s127H1 35 1376 9.0100.0 130 10 s86B4 36 1380 9.0 96.0 150 13 s55G7 37 1460 18.1 100.0 1509 s74H9 34 1335 11.3 99.7 130 10 s6E12 38 1479 13.1 90.8 390 34

TABLE 8 Molecular weights as determined by SEC-MALS for polypeptides ofthe invention and their complexes with Fab fragments of CR6261 andCR9114. Theoretical (theor) values are estimated on the basis of thesequence of the polypeptide of the invention (assuming a monomer) and anadditional contribution of approximately 10 kDa from attached glycans:The molecular weights of the Fab fragments o CR6261, CR9114 and CR8020were also determined by SEC-MALS, and were 48, 49 and 47 kDa,respectively. MW complex MW complex SEQ MW with CR6261 with CR9114 ID(kDa) (kDa) (kDa) NO: Theor Observed Theor Observed Theor Observeds127H1 35 40 39 87 74 86 83 s86B4 36 40 40 88 75 87 83 s55G7 37 40 40 9066 87 80 s74H9 34 40 41 89 72 88 83 s6E12 38 40 40 88 67 87 80

TABLE 9 Mutations created in SEQ ID NO: 156. Corresponding amino acidsin SEQ ID NO: 1 (full length, wt HA) and SEQ ID NO: 52 are alsoindicated. residue SEQ SEQ ID ID NO: Position NO: 1 156 amino acidsintroduced Set 1 337 I I E, K, V 340 I I K, R, T 352 F F D, V, Y 353 I IK, R, T 406 F S I, N, T, Y, S 409 V T A, G, I, R, T, V 413 F G I, N, S,T, Y, G 416 L S H, I, N, R, S Set 2 337 I I A, E, K, T, V 340 I I F, N,S, T, Y 352 F F A, D, I, N, S, T, V, Y 353 I I E, G, K, R, V 406 F S F,H, L, Y, S 409 V T F, I, S, T 413 F G E, K, M, V, G 416 L S I, R, S

TABLE 10 Molecular weights as determined by SEC-MALS for polypeptides ofthe invention and their complexes with Fab fragments of CR6261 andCR9114. Theoretical values (given in brackets) are estimated on thebasis of the sequence of the polypeptide of the invention (assuming atrimer) and an additional contribution of approximately 10 kDa fromattached glycans. The molecular weights of the Fab fragments of CR6261,CR9114 and CR8020 were also determined by SEC-MALS, and were 48, 49 and47 kDa, respectively. **Mw (kDa) Protein in complex with Construct NameProtein CRF9114 CRF6261 SEQ ID NO: 158 118 (120) 236 (246) 201 (255) FLHA H1N1* 210 (210) 343 (345) 396 (363) *Data included for referencepurpose **As determined from SEC MALS; theoretical values for trimericFL HA or SEQ ID NO: 158 and the trimeric FL HA or SEQ ID NO: 158 incomplex with 3 Fabs are given between brackets

TABLE 11 Polypeptides of the invention derived from SEQ ID NO: 156 andselected as described in example 11 and 12. Only residues varied in set1 and set 2 are indicated, all other esidues are identical to SEQ ID NO156. SEQ ID residue number C-terminus clone name NO: 337 340 352 353 406409 413 416 Flag-foldon-His 156 I I F I S T G S Flag-foldon-His GW1.5D10159 K K F K F T Y N GW1.5E2 158 K I Y K I T T R GW1.7H3 160 E K F T F GI N GW1.9C7 161 K I Y R T T I S GW1.8C7 162 E R F K Y V T S TCS-HisGW1.5E2 163 K I Y K I T T R GW1.9A5 164 K K F T S A Y S GW1.9E8 165 K IY K F A T N GW1.2C8 166 I T Y K S V Y N

REFERENCES

-   Alberini et al. (2009), Vaccine 27: 5998-6003.-   Bommakanti et al. (2010), PNAS 107(31): 13701-13706.-   Bommakanti et al. (2012), J Virol 86: 13434.-   Cheng et al. (2014), J. Immunol. Methods 1-13.    (doi:10.1016/j.jim.2014.07.010) Coffman et al. (2010), Immunity 33:    492.-   Devereux et al. (1984), Nucl. Acids Res. 12: 387.-   DiLillo et al. (2014), Nat Med 20, 143.-   Dopheide T A, Ward C W. (1981) J Gen Virol. 367-370-   Ekiert et al. (2009), Science 324:246.-   Ekiert et al. (2011), Science 333: 844.-   Ferguson et al. (2003), Nature 422: 428-443.-   Lorieau et al. 2010, Proc. Natl. Acad. Sci. USA, 107: 11341.-   Lu et al. (2013), www.pnas.org/cgi/doi/10.1073/pnas.1308701110.-   Mallajosyula et al (2014), www.pnas.org/cgi/doi/10    1073/pnas.1402766111.-   Parekh et al. (2012), mAbs 4: 310.-   Schnueriger et al. (2011), Molecular immunology 48: 1512.-   Steel et al. (2010), mBio 1(1): 1-9.-   Steven et al. (2004) Science 303: 1866.-   Steven et al. (2006) Science 312: 404.-   Temperton et al. (2007) Viruses 1: 105-12.-   Throsby et al. (2008), Plos One 12(3): 1-15.-   Wilson et al (1981) Nature 289: 366.

Sequences

SEQ ID NO 1: H1 Full length (A/Brisbane/59/2007)MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL  50ENSHNGKLCL LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVEKP 100NPENGTCYPG HFADYEELRE QLSSVSSFER FEIFPKESSW PNHTVTGVSA 150SCSHNGESSF YRNLLWLTGK NGLYPNLSKS YANNKEKEVL VLWGVHHPPN 200IGDQKALYHT ENAYVSVVSS HYSRKFTPEI AKRPKVRDQE GRINYYNTLL 250EPGDTIIFEA NGNLIAPRYA FALSRGFGSG IINSNAPMDK CDAKCQTPQG 300

LDFHDSNVKN LYEKVKSQLK NNAKEIGNGC FEFYHKCNDE CMESVKNGTY 500DYPKYSEESK LNREKIDGVK LESMGVYQIL AIYSTVASSL VLLVSLGAIS 550FWMCSNGSLQ CRICI 565 SEQ ID NO: 2: H1-mini2-cluster1+5+6-GCN4MKVKLLVLLC TFTATYA DTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL  50

VKSQLKNNAK EIGNGCFEFY HKCNDECMES VKNGTYDYPK YSEESKLNRE 250KIDGVKLESM GVYQILAIYS TVASSLVLLV SLGAISFWMC SNGSLQCRIC 300 I 301SEQ ID NO: 3: foldon GYIPEAPRDGQAYVRKDGEWVLLSTFLSEQ ID NO: 4: FLAG-thrombin-foidon-HISSGRDYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH SEQ ID NO: 5:MKQIEDKIESKQ SEG ID NO: 6: H1-mini2-cluster1+5+6-GCN4 without leadersequence and with FLAG-thrombin-foldon-HISDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNIPSIQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQSTATGKEGNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVSGRDYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHSEQ ID NO 7: H1 consensus sequence residue 402-418(numbering according to SEQ ID NO: 1)402 MNTQFTAVG KEFN(H/K)LE(K/R) 418 >SC09-114 VH PROTEIN (SEQ ID NO: 11)QVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGLDWMGGISPIEGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAVYFCARHGNYYYYSGMDVWGQGTTVTVSS >SC09-114 VL PROTEIN (SEG ID NO: 12)SYVLTQPPAVSGTPGQRVTISCSGSDSNIGRRSVNWYQQFPGTAPKLLIYSNDQRPSVVPDRFSGSKSGTSASLAISGLQSEDEAEYYCAAWDDSLKGAVFGGGTQLTVL >CR6261 VH PROTEIN (SEQ ID NO: 9)E V Q L V E S G A E V K K P G S S V K V S C K A S G G P F RS Y A I S W V R Q A P G Q G P E W M G G I I P I F G T T K YA P K F Q G R V T I T A D D F A G T V Y M E L S S L R S E DT A M Y Y C A K H M G Y Q V R E T M D V W G K G T T V T V SS >CR6261 VL PROTEIN (SEQ ID NO: 10)Q S V L T Q P P S V S A A P G Q K V T I S C S G S S S N I GN D Y V S W Y Q Q L P G T A P K L L I Y D N N K R P S G I PD R F S G S K S G T S A T L G I T G L Q T G D E A N Y Y C AT W D R R P T A Y V V F G G G T K L T V L G >SC08-057 VH PROTEIN (SEQ ID NO: 13)EVQLVESGGGLVQPGGSLRLSCAASGFTDSVIFMSWVRQAPGKGLECVSIIYIDDSTYYADSVKGRFTISRHNSMGTVFLEMNSLRPDDTAVYYCATESGDFGDQTGPYHYYAMDV >SC08-057 VL PROTEIN (SEQ ID NO: 14)QSALTQPASVSGSPGQSITISCTGSSGDIGGYNAVSWYQHHPGKAPKLMIYEVTSRPSGVSDRFSASRSGDTASLTVSGLQAEDEAHYYCCSFADSNILI >SC08-020 VH PROTEIN (SEQ ID NO: 17)QVQLQQSGAEVKTPGASVKVSCKASGYTFTRFGVSWIRQAPGQGLEWIGWISAYNGDTYYAOKFQARVTMTTDTSTTTAYMEMRSLRSDDTAVYYCAREPPLFYSSWSLDN >SC08-020 VL PROTEIN (SEQ ID NO: 18)EIVXTQSPGTLSLSPGERATLSCRASQSVSMNYLAWFQQKPGQAPRLLIYGASRRATGIPDRISGSGSGTDFTLTISRLEPADFAVYYCQQYGTSPRTSEQ ID NO: 52: H1-mini2-cluster1+5+6-GCN4t2MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL  50

VESQLKNNAK EIGNGCFEFY HKCNDECMES VKNGTYDYPK YSEESKLNRE 250KIDGVKLESM GVYQILAIYS TVASSLVLLV SLGAISFWMC SNGSLQCRIC 300 I 301SEQ ID NO: 53: H1-mini2-cluster1+5+6-GCN4t3MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL 50

VKSQLKNNAK EIGNGCFEFY HKCNDECMES VKNGTYDYPK YSEESKLNRE 250KIDGVKLESM GVYQILAIYS TVASSLVLLV SLGAISFWMC SNGSLQCRIC 300 I 301SEQ ID NO: 55: 127H1MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEYNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 56: 86B4MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGKEMNKIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 57: 74H9MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAFGKEMNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 53: 6E12MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAFGKEVNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 59: 55G7MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEMNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLNSLGAISFWMCSNGSLQCRICI SEQ ID NO: 60: 115A1MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGYTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQITAVGKEYNKIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 61: 71H2MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAIGKEVNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 62: 181H9MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNVPSKQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKNERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 63: 220C9MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSTQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTATGKEYNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 64: 113E7MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTATGKEINKHERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 65: s74H9DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAFGKEMNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 66: s127H1DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEYNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 67: s86B4DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGKEMNKIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 68: s55G7DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEMNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 69: s6E12DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAFGKEVNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 70: s115A1DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGYTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQITAVGKEYNKIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 71: s71H2DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAIGKEVNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 76: s181H9DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNVPSKQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKNERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 77: s220C9DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSTQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTATGKEYNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 78: s113E7DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTATGKEINKHERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 72: s74H9-longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTATGKEMNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 73: s127H1-longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEYNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 74: s86134-longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGKEHNKIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 75: s55G7-longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEMNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 144: s6E12-longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAFGKEVNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 79: s115AlongDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGYTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQITAVGKEYNKIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 80: s71H2longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAIGK

GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 81: 127H1-t2

MVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK

NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 82: 86B4-t2MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGKEMNKIERRMYQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 83: 74H9-t2MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAFGKEMNKSERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 84: 6E12-t2MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAFGKEVNKLERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 35: 55G7-t2MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEMNKLERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLNLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 86: 115A1-t2MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGYTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQITAVGKEYNKIERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 87: 71H2-t2MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAIGKEVNKSERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 88: 181H9-t2MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNVPSKQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKNERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 89: 220C9-t2MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSTQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTATGKEYNKLERRNKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 90: 113E7-t2MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTATGKEINKHERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 91: s127H1-t2

GAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGK

GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHSEQ ID NO: 92: s86B4-t2DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGKEMNKIERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECNESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 93: s74H9-t2DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAFGKEMNKSERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGEPGHHHHHHSEQ ID NO: 94: s6E12-t2DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAFGKEVNKLERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 95: s55G7-t2DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEMNKLERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 96: s115A1-t2DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGYTEGGWTDMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQITAVGKEYNKIERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 97: s71H2-t2DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAIGKEVNKSERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 93: s181H9-t2DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNVPSKQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKNERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 99: s220C9-t2DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSTQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTATGKEYNKLERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 100: S113E7-t2DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTATGKEINKHERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 101: s127H1-t2long

GAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEYNKSERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 102: s86B4-t2longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGKEMNKIERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 103: s74H9-t2longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAFGKEMNKSERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 104: s6E12-t2longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAFGKEVNKLERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 105: s55G7-t2longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEMNKLERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 106: s115A1-t2longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGYTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQITAVGKEYNKIERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 107: s71H2-t2longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAIGKEVNKSERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 108: s181H9-t2longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNVPSKQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKNERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 109: s220C9-t2longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSTQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTATGKEYNKLERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 110: s113E7-t2longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTATGKEINKHERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 111: 127H1-t3

MVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEYNKSRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 112: 86B4-t3

MVTGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGKEMNKIRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 113: 74H9-t3MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAFGKEMNKSRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 114: 6E12-t3MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAFGKEVNKLRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 115: 55G7-t3MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEMNKLRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 116: 115A1-t3MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGYTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQITAVGKEYNKIRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 117: 71H2-t3MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAIGKEVNKSRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 118: 181H9-t3MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNVPSKQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKNRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 119: 220C9-t3MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSTQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTATGKEYNKLRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 120: 113E7-t3MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTATGKEINKHRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 121: s127H1-t3

GAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEYNKSRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 122: s86B4-t3

GAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGKEMNKIRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 123: s74H9-t3DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGATAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAFGKEMNKSRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECNESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 124: s6E12-t3DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTOLTAFGKEVNKLRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 125: s55G7-t3DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEMNKLRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 126: s115A1-t3DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGYTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQITAVGKEYNKIRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 127: s71H2-t3DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAIGKEVNKSRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 128: s181H9-t3DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNVPSKQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKNRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 129: s220C9-t3DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSTQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTATGKEYNKLRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 130: s113E7-t3DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTATGKEINKHRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHHSEQ ID NO: 131: s127H1-t3long

GAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEYNKSRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 132: s86B4-t3longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGKEMNKIRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 133: s74H9-t3longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAFGKEMNKSRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 134: s6E12-t3longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAFGKEVNKLRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 135: s55G7-t3longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGKEMNKLRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 136: s115A1-t3longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGYTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQITAVGKEYNKIRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 137: s71H2-t3longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAIGKEVNKSRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 138: s181H9-t3longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNVPSKQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKNRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 139: s220C9-t3longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSTQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTATGKEYNKLRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 140: s113E7-t3longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGATAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTATGKEINKHRMKQIEDKIEEIESKQKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 141: s181H9longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNVPSKQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKNERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 142: s220C9longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSTQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTATGKEYNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 143: s113E7longDTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTATGKEINKHERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGSEQ ID NO: 149: smH1 Cali3964-55G7MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDGGGGKYVCSTKLRLATGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQYTAIGKEMNHLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGRSLVPRGSPGHHHHHH SEQ ID NO: 150: smH1 Cali3964-86B4MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDGGGGKYVCSTKLRLATGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAIGKEMNHIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGRSLVPRGSPGHHHHHH SEQ ID NO: 151: smH1 Cali3964-127H1MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDGGGGKYVCSTKLRLATGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQYTAIGKEYNHSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGRSLVPRGSPGHHHHHH SEQ ID NO: 152: _smH1 Cali3964-55G7-t2MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDGGGGKYVCSTKLRLATGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQYTAIGKEMNHLERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGRSLVPRGSPGHHHHHH SEQ ID NO: 153: _smH1 Cali3964-86B4-t2MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDGGGGKYVCSTKLRLATGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAIGKEMNHIERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGRSLVPRGSPGHHHHHH SEQ ID NO: 154: smH1 Cali3964-127H1-t2MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDGGGGKYVCSTKLRLATGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQYTAIGKEYNHSERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGRSLVPRGSPGHHHHHH SEQ ID NO: 155: mH1 Cali3964-127H1-t2MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDGGGGKYVCSTKLRLATGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQYTAIGKEYNHSERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICISEQ ID NO: 156: sH1-mini2-cluster1+5+6-GCN4t2 without leadersequence and with FLAG-foldon-HIS

EIGNGCFEFY HKCNDECMES VKNGTYDYPK YSEESKLNRE KIDGVSGRDYKDDDDKPGSG YIPEAPRDGQ AYVRKDGEWV LLSTFLGHHH HHHSEQ ID NO: 157: H1 mini-HA GW1.5E2MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLLENGGGGKYVC SAKLRMVTGL RNKPSIQSQG LFGAIAGYKE GGWTGMVDGWYGYHHQNEQG SGYAADQKST QNAINGITNK VNSVIEKMNT QITATGKETNKRERRMKQIE DKIEEIESKI WCYNAELLVL LENERTLDFH DSNVKNLYEKVKSQLKNNAK EIGNGCFEFY HKCNDECMES VKNGTYDYPK YSEESKLNREKIDGVKLESM GVYQILAIYS TVASSLVLLV SLGAISFWMC SNGSLQCRICISEQ ID NO: 158 sH1 mini-HA GW1.5E2-FFH (#5145)       DTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL ENGGGGKYVCSAKLRMVTGL RNKPSIQSQG LFGAIAGYKE GGWTGMVDGW YGYHHQNEQGSGYAADQKST QNAINGITNK VNSVIEKMNT QITATGKETN KRERRMKQIEDKIEEIESKI WCYNAELLVL LENERTLDFH DSNVKNLYEK VKSQLKNNAKEIGNGCFEFY HKCNDECMES VKNGTYDYPK YSEESKLNRE KIDGVSGRDYKDDDDKPGSG YIPEAPRDGQ AYVRKDGEWV LLSTFLGHHH HHH

1-15. (canceled)
 16. An influenza hemagglutinin stem domain polypeptidecomprising the amino acid sequence: (SEQ ID NO: 146)DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMV TGLRNX ₁PSX₂QSQGLFGAIAGX ₃ X ₄EGGWTGMVDGWYGYHHQNEQGSGYA ADQKSTQNAINGITNKVNSVIEKX₅NTQX ₆TAX ₇GKEX ₈NKX ₉ERRMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDG,

wherein X₁ is an amino acid selected from the group consisting of E, I,K, V, A, and T; X₂ is an amino acid selected from the group consistingof I, K, R, T, F, N, S and Y; X₃ is an amino acid selected from thegroup consisting of D, F, V, Y, A, I, N, S, and T; X₄ is an amino acidselected from the group consisting of I, K, R, T, E, G and V; X₅ is anamino acid selected from the group consisting of, M, E, K, V, R, T; X₆is an amino acid selected from the group consisting of F, I, N, S, T, Y,H, and L; X₇ is an amino acid selected from the group consisting of A,G, I, R, T, V, F, and S; X₈ is an amino acid selected from the groupconsisting of F, I, N, S, T, Y, G, E, K, M and V; and X₉ is an aminoacid selected from the group consisting of H, I, L, N, R, and S.
 17. Theinfluenza hemagglutinin stem domain polypeptide according to claim 16,wherein the polypeptide selectively binds to the antibodies CR6261and/or CR9114.
 18. A nucleic acid molecule encoding the polypeptide ofclaim
 16. 19. A vector comprising the nucleic acid molecule of claim 18.20. A composition comprising the polypeptide of claim 16 and apharmaceutically acceptable carrier.
 21. A composition comprising thenucleic acid molecule according to claim 18 and a pharmaceuticallyacceptable carrier.
 22. A method of inducing an immune response againstan influenza virus in a subject in need thereof, the method comprisingadministering to the subject in need thereof the polypeptide accordingto claim
 16. 23. A method of inducing an immune response against aninfluenza virus in a subject in need thereof, the method comprisingadministering to the subject in need thereof the nucleic acid moleculeaccording to claim
 18. 24. A method of inducing an immune responseagainst an influenza virus in a subject in need thereof, the methodcomprising administering to the subject in need thereof the vectoraccording to claim
 19. 25. A composition comprising the vector accordingto claim 19 and a pharmaceutically acceptable carrier.
 26. The influenzahemagglutinin stem domain polypeptide according to claim 16, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:81.